Antagonists of neuropilin receptor function and use thereof

ABSTRACT

The present invention relates to antagonists of neuropilin receptor fuction and use thereof in the treatment of cancer, particularly metastatic cancer, and angiogenic diseases.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The work described herein was supported, in part, by National Instituteof Health grants CA37392 and CA45548. The U.S. Government has certainrights to the invention.

FIELD OF THE INVENTION

The present invention relates to antagonists of neuropilin receptorfunction and use thereof in the treatment of cancer, particularlymetastatic cancer, and angiogenic diseases.

BACKGROUND OF THE INVENTION

Cancer, its development and treatment is a major health concern. Thestandard treatments available are few and directed to specific types ofcancer, and provide no absolute guarantee of success. Most treatmentsrely on an approach that involves killing off rapidly growing cells inthe hope that rapidly growing cancerous cells will succumb, either tothe treatment, or at least be sufficiently reduced in numbers to allowthe body's system to eliminate the remainder. However most, of thesetreatments are non-specific to cancer cells and adversely effectnon-malignant cells. Many cancers although having some phenotyperelationship are quite diverse. Yet, what treatment works mosteffectively for one cancer may not be the best means for treatinganother cancer. Consequently, an appreciation of the severity of thecondition must be made before beginning many therapies. In order to mosteffective, these treatments require not only an early detection of themalignancy, but an appreciation of the severity of the malignancy.Currently, it can be difficult to distinguish cells at a molecular levelas it relates to effect on treatment. Thus, methods of being able toscreen malignant cells and better understand their disease state aredesirable.

While different forms of cancer have different properties, one factorwhich many cancers share is that they can metastasize. Until such timeas metastasis occurs, a tumor, although it may be malignant, is confinedto one area of the body. This may cause discomfort and/or pain, or evenlead to more serious problems including death, but if it can be located,it may be surgically removed and, if done with adequate care, betreatable. However, once metastasis sets in, cancerous cells haveinvaded the body and while surgical resection may remove the parenttumor, this does not address other tumors. Only chemotherapy, or someparticular form of targeting therapy, then stands any chance of success.

The process of tumor metastasis is a multistage event involving localinvasion and destruction of intercellular matrix, intravasation intoblood vessels, lymphatics or other channels of transport, survival inthe circulation, extravasation out of the vessels in the secondary siteand growth in the new location (Fidler, et al., Adv. Cancer Res. 28,149-250 (1978), Liotta, et al., Cancer Treatment Res. 40, 223-238(1988), Nicolson, Biochim. Biophy. Acta 948, 175-224 (1988) and Zetter,N. Eng. J. Med. 322, 605-612 (1990)). Success in establishing metastaticdeposits requires tumor cells to be able to accomplish these stepssequentially. Common to many steps of the metastatic process is arequirement for motility. The enhanced movement of malignant tumor cellsis a major contributor to the progression of the disease towardmetastasis. Increased cell motility has been associated with enhancedmetastatic potential in animal as well as human tumors (Hosaka, et al.,Gann 69, 273-276 (1978) and Haemmerlin, et al., Int. J. Cancer 27,603-610 (1981)).

Identifying factors that are associated with onset of tumor metastasisis extremely important. In addition, to using such factors for diagnosisand prognosis, those factors are an important site for identifying newcompounds that can be used for treatment and as a target for treatmentidentifying new modes of treatment such as inhibition of metastasis ishighly desirable.

Tumor angiogenesis is essential for both primary tumor expansion andmetastatic tumor spread, and angiogenesis itself requires ECMdegradation (Blood et al., Biochim. Biophys. Acta 1032:89-118 (1990)).Thus, malignancy is a systemic disease in which interactions between theneoplastic cells and their environment play a crucial role duringevolution of the pathological process (Fidler, I. J., Cancer MetastasisRev. 5:29-49 (1986)).

There is mounting evidence that VEGF may be a major regulator ofangiogenesis (reviewed in Ferrara, et al., Endocr. Rev., 13, 18-32(1992); Klagsbrun, et al., Curr. Biol., 3, 699-702 (1993); Ferrara, etal., Biochem. Biophjs. Res. Commun., 161, 851-858 (1989)). VEGF wasinitially purified from the conditioned media of folliculostellate cells(Ferrara, et al., Biochem. Biophjs. Res. Commun., 161, 851-858 (1989))and from a variety of tumor cell lines (Myoken, et al., Proc. Natl.Acad. Sci. USA, 88:5819-5823 (1991); Plouet, et al., EMBO. J.,8:3801-3806 (1991)). VEGF was found to be identical to vascularpermeability factor, a regulator of blood vessel permeability that waspurified from the conditioned medium of U937 cells at the same time(Keck, et al., Science, 246:1309-1312 (1989)). VEGF is a specificmitogen for endothelial cells (EC) in vitro and a potent angiogenicfactor in vivo. The expression of VEGF is up-regulated in tissueundergoing vascularization during embryogenesis and the femalereproductive cycle (Brier, et al., Development, 114:521-532 (1992);Shweiki, et al., J. Clin. Invest., 91:2235-2243 (1993)). High levels ofVEGF are expressed in various types of tumors, but not in normal tissue,in response to tumor-induced hypoxia (Shweiki, et al., Nature359:843-846 (1992); Dvorak et al., J. Exp. Med., 174:1275-1278 (1991);Plate, et al., Cancer Res., 53:5822-5827; Ikea, et al., J. Biol. Chem.,270:19761-19766 (1986)). Treatment of tumors with monoclonal antibodiesdirected against VEGF resulted in a dramatic reduction in tumor mass dueto the suppression of tumor angiogenesis (Kim, et al., Nature,382:841-844 (1993)). VEGF appears to play a principle role in manypathological states and processes related to neovascularization.Regulation of VEGF expression in affected tissues could therefore be keyin treatment or prevention of VEGF inducedneovascularization/angiogenesis.

VEGF exists in a number of different isoforms that are produced byalternative splicing from a single gene containing eight exons (Ferrara,et al., Endocr. Rev., 13:18-32 (1992); Tischer, et al., J. Biol. Chem.,806:11947-11954 (1991); Ferrara, et al., Trends Cardio Med., 3:244-250(1993); Polterak, et al., J. Biol. Chem., 272:7151-7158 (1997)). HumanVEGF isoforms consists of monomers of 121, 145, 165, 189, and 206 aminoacids, each capable of making an active homodimer (Polterak et al., J.Biol. Chem., 272:7151-7158 (1997); Houck, et al., Mol. Endocrinol.,8:1806-1814 (1991)). The VEGF₁₂₁ and VEGF₁₆₅ isoforms are the mostabundant. VEGF₁₂₁ is the only VEGF isoforms that does not bind toheparin and is totally secreted into the culture medium. VEGF₁₆₅ isfunctionally different than VEGF₁₂₁ in that it binds to heparin and cellsurface heparin sulfate proteoglycans (HSPGs) and is only partiallyreleased into the culture medium (Houck, et al., J. Biol. Chem.,247:28031-28037 (1992); Park, et al., Mol. Biol. Chem., 4:1317-1326(1993)). The remaining isoforms are entirely associated with cellsurface and extracellular matrix HSPGs (Houck, et al., 1 Biol. Chem.,247:28031-28037 (1992); Park, et al., Mol. Biol. Chem., 4:1317-1326(1993)).

VEGF receptor tyrosine kinases, KDR/Flk-1 and/or Flt-1, are mostlyexpressed by EC (Terman, et al., Biochem. Biophys. Res. Commun.,187:1579-1586 (1992); Shibuya, et al., Oncogene, 5:519-524 (1990); DeVries, et al., Science, 265:989-991 (1992); Gitay-Goran, et al., J.Biol. Chem., 287:6003-6096 (1992); Jakeman, et al., J. Clin. Invest.,89:244-253 (1992)). It appears that VEGF activities such asmitogenicity, chemotaxis, and induction of morphological changes aremediated by KDR/Flk-1 but not Flt-1, even though both receptors undergophosphorylation upon binding of VEGF (Millauer, et al., Cell, 72:835-846(1993); Waltenberger, et al., J. Biol. Chem., 269:26988-26995 (1994);Seetharam, et al., Oncogene, 10:135-147 (1995); Yoshida, et al., GrowthFactors, 7:131-138 (1996)). Recently, Soker et al., identified a newVEGF receptor which is expressed on EC and various tumor-derived celllines such as breast cancer-derived MDA-MB-231 (231) cells (Soker, etal., J. Biol. Chem., 271:5761-5767 (1996)). This receptor requires theVEGF isoform to contain the portion encoded by exon 7. For example,although both VEGF₁₂₁ and VEGF₁₆₅R bind to KDR/Flk-1 and Flt-1, onlyVEGF₁₆₅ binds to the new receptor. Thus, this is an isoform-specificreceptor and has been named the VEGF₁₆₅ receptor (VEGF₁₆₅R). It willalso bind the 189 and 206 isoforms. VEGF₁₆₅R has a molecular mass ofapproximately 130 kDa, and it binds VEGF₁₆₅ with a Kd of about 2×10⁻¹⁰M,compared with approximately 5×10⁻¹²M for KDR/Flk-1. Instructure-function analysis, it was shown directly that VEGF₁₆₅ binds toVEGF₁₆₅R via its exon 7-encoded domain which is absent in VEGF₁₂₁(Soker, et al., J Biol. Chem., 271:5761-5767 (1996)). However, thefunction of the receptor was unclear.

Identifying the alterations in gene expression which are associated withmalignant tumors, including those involved in tumor progression andangiogenesis, is clearly a prerequisite not only for a fullunderstanding of cancer, but also to develop new rational therapiesagainst cancer.

A further problem arises, in that the genes characteristic of cancerouscells are very often host genes being abnormally expressed. It is quiteoften the case that a particular protein marker for a given cancer whileexpressed in high levels in connection with that cancer is alsoexpressed elsewhere throughout the body, albeit at reduced levels.

The current treatment of angiogenic diseases is inadequate. Agents whichprevent continued angiogenesis, e.g, drugs (TNP-470), monoclonalantibodies, antisense nucleic acids and proteins (angiostatin andendostatin) are currently being tested. See, Battegay, J. Mol. Med., 73,333-346 (1995); Hanahan et al., Cell, 86, 353-364 (1996); Folkman, N.Engl. J. Med., 333, 1757-1763 (1995). Although preliminary results withthe antiangiogenic proteins are promising, there is still a need foridentifying genes encoding ligands and receptors involved inangiogenesis for the development of new antiangiogenic therapies.

SUMMARY OF THE INVENTION

We have isolated a cDNA encoding the VEGF₁₆₅ R gene (SEQ ID NO: 1) andhave deduced the amino acid sequence of the receptor (SEQ ID NO:2) Wehave discovered that this novel VEGF receptor is structurally unrelatedto Flt-1 or KDR/Flk-1 and is expressed not only by endothelial cells butby non-endothelial cells, including surprisingly tumor cells.

In ascertaining the function of the VEGF₁₆₅R we have further discoveredthat this receptor has been identified as a cell surface mediator ofneuronal cell guidance and called neuropilin-1. Kolodkin et al., Cell90:753-762 (1997). We refer to the receptor as VEGF₁₆₅R/NP-1 or NP-1.

In addition to the expression cloning of VEGF₁₆₅R/NP-1 cDNA we isolatedanother human cDNA clone whose predicted amino acid sequence was 47%homologous to that of VEGF₁₆₅R/NP-1 and over 90% homologous to ratneuropilin-2 (NP-2) which was recently cloned (Kolodkin, et al., Cell90, 753-762 (1997)). Our results indicate that VEGF₁₆₅R/NP-1 and NP-2are expressed by both endothelial and tumor cells. (FIG. 19) We haveshown that endothelial cells expressing both KDR and VEGF₁₆₅R/NP-1respond with increased chemotaxis towards VEGF₁₆₅, not VEGF₁₂₁, whencompared to endothelial cells expressing KDR alone. While not wishing tobe bound by theory, we believe that VEGF₁₆₅R/NP-1 functions inendothelial cells to mediate cell motility as a co-receptor for KDR.

We have also shown in the Boyden chamber motility assay that VEGF₁₆₅stimulates 231 breast carcinoma cell motility in a dose-response manner(FIG. 15A). VEGF₁₂₁ had no effect motility of these cells (FIG. 15B).Since tumor cells such as, 231 cells, do not express the VEGF receptors,KDR or Flt-1, while not wishing to be bound by theory, we believe thattumor cells are directly responsive to VEGF₁₆₅ via VEGF₁₆₅R/NP-1.

We have also analyzed two variants of Dunning rat prostate carcinomacells, AT2.1 cells, which are of low motility and low metastaticpotential, and AT3.1 cells, which are highly motile, and metastatic.Cross-linking and Northern blot analysis show that AT3.1 cells expressabundant VEGF₁₆₅R/NP-1, capable of binding VEGF₁₆₅, while AT2.1 cellsdon't express VEGF₁₆₅R/NP-1 (FIG. 18). Immunostaining of tumor sectionsconfirmed the expression of VEGF₁₆₅R/NP-1 in AT3.1, but not AT2.1 tumors(FIG. 17). Additionally, immunostaining showed that in subcutaneousAT3.1 and PC3 tumors, the tumor cells expressing VEGF₁₆₅R/NP-1 werefound preferentially at the invading front of the tumor/dermis boundary(FIG. 17). Furthermore, stable clones of AT2.1 cells overexpressingVEGF₁₆₅R/NP-1 had enhanced motility in the Boyden chamber assay. Theseresults indicate that neuropilin expression on tumor cells is associatedwith the motile, metastatic phenotype and angiogenesis, and thus is animportant target for antiangiogenic and anticancer therapy.

The present invention relates to antagonists of neuropilin (NP) receptorfunction that can be use to inhibit metastasis and angiogenesis.Antagonists of invention can block the receptor preventing ligandbinding, disrupt receptor function, or inhibit receptor occurrence.Specific antagonists include, for example, compounds that bind to NP-1or NP-2 and antibodies that specifically binds the receptor at a regionthat inhibits receptor function. For example, one can add an effectiveamount of a compound that binds to NP-1 to disrupt receptor fuction andthus inhibit metastasis.

We have surprisingly discovered that members of thesemaphorin/collapsins family are not only inhibitors of neuronalguidance but also inhibitors of endothelial and tumor cell motility incells that express neuropilin. Accordingly, preferred antagonistsinclude members of the semaphorin/collapsins family or fragments thereofthat bind NP and have VEGF antagonist activity as determined, forexample, by the human umbilical vein endothelial cell (HUVEC)proliferation assay using VEGF₁₆₅ as set forth in Soker et al., J. Biol.Chem. 272, 31582-31588 (1997). Preferably, the semaphorin/collapsin hasat least a 25% reduction in HUVEC proliferation, more preferably a 50%reduction, even more preferably a 75% reduction, most preferably a 95%reduction.

VEGF antagonist activity of the semaphorin/collapsin may also bedetermined by inhibition of binding of labeled VEGF₁₆₅ to VEGF₁₆₅R asdisclosed in Soker et al., J. Biol. Chem. 271, 5761-5767 (1996)) or toPAE/NP cells. Preferably, the portion inhibits binding by at least 25%,more preferably 50%, most preferably 75%.

In accordance with the present invention, neuropilin antagonists, ornucleic acids, e.g., DNA or RNA, encoding such antagonists, are usefulas inhibitors of VEGF and NP function and can be used to treat diseases,disorders or conditions associated with VEGF and NP expression. Theantagonists can be used alone or in combination with other anti-VEGFstrategies including, for example, those that antagonize VEGF directly(e.g. anti-VEGF antibodies, soluble VEGF receptor extracellulardomains), or antagonize VEGF receptors (e.g. anti-KDR antibodies, KDRkinase inhibitors, dominant-negative VEGF receptors) (Presta L G, etal., Cancer Res. 57: 4593-4599 (1997), Kendall R L, et al., (1996)Biochem. Biophys. Res. Commun. 226: 324-328, Goldman C K, et al., (1998)Proc. Natl. Acad. Sci. USA 95: 8795-8800, Strawn L M, et al., (1996)Cancer Res. 56: 3540-3545, Zhu Z, et al., (1998). Cancer Res. 58:3209-3214, Witte L, et al., (1998). Cancer Metastasis Rev. 17: 155-161.)

Diseases, disorders, or conditions, associated with VEGF, include, butare not limited to retinal neovascularization, hemagiomas, solid tumorgrowth, leukemia, metastasis, psoriasis, neovascular glaucoma, diabeticretinopathy, rheumatoid arthritis, endometriosis, muscular degeneration,osteoarthritis, and retinopathy of prematurity (ROP).

In another embodiment, one can use isolated VEGF₁₆₅R/NP-1 or NP-2 orcells expressing these receptors in assays to discover compounds thatbind to or otherwise interact with these receptors in order to discoverNP antagonists that can be used to inhibit metastasis and/orangiogenesis.

Other aspects of the invention are disclosed infra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows purification of VEGF₁₆₅R From 231 Cells. ¹²⁵I-VEGF₁₆₅ (5ng/ml) was bound and cross-linked to receptors on 231 cells and analyzedby SDS PAGE and autoradiography (lane 1). VEGF₁₆₅R was purified by Con Aand VEGF₁₆₅ affinity column chromatography and analyzed by SDS-PAGE andsilver stain (lane 2). Two prominent bands were detected (arrows) andN-terminally sequenced separately. Their N-terminal 18 amino acidsequences are shown to the right of the arrows. The published N-terminalsequences of human and mouse neuropilin (Kawakami et al., J. Neurobiol.,29, 1-17 (1995); He and Tessier-Lavigne, Cell 90, 739-751 1997) areshown below (SEQ ID NOS: 5 and 6).

FIGS. 2A and 2B show isolation of VEGF₁₆₅R cDNA by Expression Cloning.Photomicrographs (dark field illumination) of COS 7 cells binding¹²⁵I-VEGF₁₆₅. ¹²⁵I-VEGF₁₆₅ was bound to transfected COS 7 cells whichwere then washed, fixed, and overlayed with photographic emulsion thatwas developed as described in the example, infra.

FIG. 2A shows COS 7 cells were transfected with a primary plasmid pool(#55 of the 231 cell library) representing approximately 3×10³ clonesand one COS 7 cell binding ¹²⁵I-VEGF₁₆₅ in the first round of screeningis shown.

FIG. 2 shows several COS 7 cells transfected with a single positive cDNAclone (A2) binding ¹²⁵I-VEGF₁₆₅ after the third round of screening.

FIG. 3 shows the Deduced Amino Acid Sequence of Human VEGF₁₆₅R/NP-1 (SEQID NO:3). The deduced 923 amino acid sequence of the open reading frameof VEGF₁₆₅R/NP-1, clone A2 (full insert size of 6.5 kb) is shown. Theputative signal peptide sequence (amino acids 1-21) and the putativetransmembrane region (amino acids 860-883) are in boxes. The amino acidsequence obtained by N-terminal amino acid sequencing (FIG. 3, aminoacids 22-39) is underlined. The arrow indicates where the signal peptidehas been cleaved and removed, based on comparison of the N-terminalsequence of purified VEGF₁₆₅R/NP-1 and the cDNA sequence. The sequenceof human VEGF₁₆₅R/NP-1 reported here differs from that reported by He etal. (He and Tessier-Lavigne, Cell 90, 739-751 (1997)) in that we findLys₂₆ rather than Glu₂₆, and Asp₈₅₅ rather than Glum. Lys₂₆ and Asp₈₅₅are found, however, in mouse and rat VEGF₁₆₅R/NP-1 (Kwakami et al., J.Neurobiol. 29, 1-17 (1995); He and Tessier-Lavigne, Cell 90, 739-751(1997)).

FIGS. 4A and 4B show the Comparison of the Deduced Amino Acid Sequenceof Human VEGF₁₆₅R/NP-1 (SEQ ID NO:2) and NP-2 (SEQ ID NO:4). The deducedopen reading frame amino acid sequences of VEGF₁₆₅R/NP-1 and NP-2 arealigned using the DNASIS program. Amino acids that are identical in bothopen reading frames are shaded. The overall homology between the twosequences is 43%.

FIG. 5 shows a Northern Blot Analysis of VEGF₁₆₅R/NP-1 Expression inHuman EC and Tumor-Derived Cell Lines. Total RNA samples prepared fromHUVEC (lane 1) and tumor-derived breast carcinoma, prostate carcinomaand melanoma cell lines as indicated (lanes 2-8) were resolved on a 1%agarose gel and blotted onto a GeneScreen nylon membrane. The membranewas probed with ³²P-labeled VEGF₁₆₅R/NP-1 cDNA and exposed to X-rayfilm. Equal RNA loading was demonstrated by ethydium bromide staining ofthe gel prior to blotting. A major species of VEGF₁₆₅R/NP-1 mRNA ofapproximately 7 kb was detected in several of the cell lines.

FIG. 6 shows a Northern Blot Analysis of VEGF₁₆₅R/NP-1 and KDR mRNA inAdult Human Tissues. A pre-made Northern blot membrane containingmultiple samples of human mRNA (Clonetech) was probed with ³²P-labeledVEGF₁₆₅R/NP-1 cDNA (top) as described in FIG. 5, and then stripped andreprobed with ³²P-labeled KDR cDNA (bottom).

FIGS. 7A and 7B show a Scatchard Analysis of VEGF₁₆₅ binding toVEGF₁₆₅R/NP-1. FIG. 7A. Increasing amounts of ¹²⁵I-VEGF₁₆₅ (0.1-50ng/ml) were added to subconfluent cultures of PAE cells transfected withhuman VEGF₁₆₅R/NP-1 cDNA (PAE/NP-1 cells) in 48 well dishes.Non-specific binding was determined by competition with a 200-foldexcess of unlabeled VEGF₁₆₅. After binding, the cells were washed, lysedand the cell-associated radioactivity was determined using a γ counter.

FIG. 7B. The binding data shown in FIG. 7A were analyzed by the methodof Scatchard, and a best fit plot was obtained with the LIGAND program(Munson and Rodbard, 1980). PAE/NP-1 cells express approximately 3×10⁵VEGF₁₆₅ binding sites per cell and bind ¹²⁵I-VEGF₁₆₅ with a K_(d) of3.2×10⁻¹⁰ M.

FIG. 8 shows cross-linking of VEGF₁₆₅ and VEGF₁₂₁ to PAE cellsExpressing VEGF₁₆₅R/NP-1 and/or KDR. ¹²⁵I-VEGF₁₆₅ (5 ng/ml) (lanes 1-6)or ¹²⁵I-VEGF₁₂₁ (10 ng/ml) (lanes 7-10) were bound to subconfluentcultures of HUVEC (lane 1), PC3 (lane 2), PAE (lanes 3 and 7), a cloneof PAE cells transfected with human VEGF₁₆₅R/NP-1 cDNA (PAE/NP-1) (lanes4 and 8), a clone of PAE cells transfected with KDR (PAE/KDR) (lanes 5and 9), and a clone of PAE/KDR cells transfected with humanVEGF₁₆₅R/NP-1 cDNA (PAE/KDR/NP-1) (lanes 6 and 10).

The binding was carried out in the presence of 1 μg/ml heparin. At theend of a 2 hour incubation, each ¹²⁵I-VEGF isoform was chemicallycross-linked to the cell surface. The cells were lysed and proteins wereresolved by 6% SDS-PAGE. The polyacrylamide gel was dried and exposed toX-ray film. Solid arrows denote radiolabeled complexes containing¹²⁵I-VEGF and KDR, open arrows denote radiolabeled complexes containing¹²⁵I-VEGF and VEGF₁₆₅R/NP-1.

FIG. 9 shows cross linking of VEGF₁₆₅ to PAE/KDR Cells Co-expressingVEGF₁₆₅R/NP-1 Transiently. PAE/KDR cells were transfected with pCPhygroor pCPhyg-NP-1 plasmids as described in “Experimental Procedures”, andgrown for 3 days in 6 cm dishes. ¹²⁵I-VEGF₁₆₅ (5 ng/ml) was bound andcross linked to parental PAE/KDR cells (lane 1), to PAE/KDR cellstransfected with pCPhygro vector control (V) (lane 2), to PAE/KDR cellstransfected with pCPhyg-VEGF₁₆₅R/NP-1 plasmids (VEGF₁₆₅R/NP-1) (lane 3),and to HUVEC (lane 4).). The binding was carried out in the presence of1 μg/ml heparin. The cells were lysed and proteins were resolved by 6%SDS-PAGE as in FIG. 8. Solid arrows denote radiolabeled complexescontaining ¹²⁵I-VEGF₁₆₅ and KDR. Open arrows denote radiolabeledcomplexes containing ¹²⁵I-VEGF₁₆₅ and VEGF₁₆₅R/NP-1.

FIG. 10 shows inhibition of ¹²⁵I-VEGF₁₆₅ binding to VEGF₁₆₅R/NP-1interferes with its binding to KDR. ¹²⁵I-VEGF₁₆₅ (5 ng/ml) was bound tosubconfluent cultures of PAE transfected with human VEGF₁₆₅R/NP-1 cDNA(PAE/NP-1) (lanes 1 and 2), PAE/KDR cells (lanes 3 and 4), and PAE/KDRcells transfected with human VEGF₁₆₅R/NP-1 cDNA (PAE/KDR/NP-1) (lanes 5and 16) in 35 mm dishes. The binding was carried out in the presence(lanes 2, 4, and 6) or the absence (lanes 1, 3, and 5) of 25 μg/mlGST-Ex 7+8. Heparin (1 μg/ml) was added to each dish. At the end of a 2hour incubation, ¹²⁵I-VEGF₁₆₅ was chemically cross-linked to the cellsurface. The cells were lysed and proteins were resolved by 6% SDS-PAGEas in FIG. 9. Solid arrows denote radiolabeled complexes containing¹²⁵I-VEGF₁₆₅ and KDR, open arrows denote radiolabeled complexescontaining ¹²⁵I-VEGF₁₆₅ and VEGF₁₆₅R/NP-1.

FIGS. 11A-C show a model for VEGF₁₆₅R/NP-1 modulation of VEGF₁₆₅ Bindingto KDR. 11A.Cells expressing KDR alone. 11B.Cells co-expressing KDR andVEGF₁₆₅R/NP-1. 11C.Cells co-expressing KDR and VEGF₁₆₅R/NP-1 in thepresence of GST-Ex 7+8 fusion protein.

A single KDR receptor or a KDR-VEGF₁₆₅R/NP-1 pair is shown in topportion. An expanded view showing several receptors is shown in thebottom portion. VEGF₁₆₅ binds to KDR via exon 4 and to VEGF₁₆₅R/NP-1 viaexon 7 (Keyt et al. J. Biol. Chem. 271, 5638-5646 (1996b); Soker et al.,J. Biol. Chem. 271, 5761-5767 (1996)). A rectangular VEGF₁₆₅ moleculerepresents a suboptimal conformation that doesn't bind to KDRefficiently while a rounded VEGF₁₆₅ molecule represents one that fitsbetter into a binding site. In cells expressing KDR alone, VEGF₁₆₅ bindsto KDR in a sub-optimal manner. In cells co-expressing KDR andVEGF₁₆₅R/NP-1, the binding efficiency of VEGF₁₆₅ to KDR is enhanced. Itmay be that the presence of VEGF₁₆₅R/NP-1 increases the concentration ofVEGF₁₆₅ on the cell surface, thereby presenting more growth factor toKDR. Alternatively, VEGF₁₆₅R/NP-1 may induce a change in VEGF₁₆₅conformation that allows better binding to KDR, or both might occur. Inthe presence of GST-Ex 7+8, VEGF₁₆₅ binding to VEGF₁₆₅R/NP-1 iscompetitively inhibited and its binding to KDR reverts to a sub-optimalmanner.

FIG. 12 shows the human NP-2 amino acid sequence (SEQ ID NO:4).

FIGS. 13A, 13B and 13C show the human NP-2 DNA sequence (SEQ ID NO:3).

FIGS. 14A, 14B, 14C, 14D, 14E and 14F show the nucleotide (SEQ ID NO:1)and amino acid sequences (SEQ ID NO:2) of VEGF₁₆₅R/NP-1.

FIGS. 15A and 15B show VEGF₁₆₅ stimulation of MDA MB 231 cell motility.(FIG. 15A) Dose response of VEGF₁₆₅ motility activity. (FIG. 15B) BothVEGF₁₆₅ and bFGF stimulate motility but VEGF₁₂₁ does not.

FIGS. 16A, 16B and 16C show motility and neuropilin-1 expression ofDunning rat prostate carcinoma cell lines AT3-1 (high motility, highmetastatic potential) and AT2.1 (low motility, low metastatic potential)cells. (FIG. 16A) AT3.1 cells are more motile than AT2.1 cells in aBoyden chamber assay. 125I-VEGF₁₆₅ cross-links neuropilin-1 on AT3.1cells but does not cross-link to AT2.1 cells. (FIG. 16C) AT3.1 but notAT2.1 cells express neuropilin-1, while both cell types express VEGF.

FIGS. 17A, 17B and 17C show immunostaining of (FIG. 17A) a PC3subcutaneous tumor in a nude mouse, (FIG. 17B) an AT3.1 tumor in a rat,(FIG. 17C) an AT2.1 tumor in rat with anti-neuropilin-1 antibodies.Neuropilin immunostaining is preferentially associated with PC3 andAT3.1 tumor cells at the tumor/dermis boundary. Some of these cellscluster around blood vessels. AT2.1 cells do not express neuropilin-1.

FIGS. 18A and 18B show overexpression of neuropilin-1 in AT2.1 cells.(FIG. 18A) Western blot, (FIG. 18B) motility activity. Three AT2.1clones (lanes 4, 5, 6) express higher amounts of neuropilin-1 proteinand are more motile compared to parental AT2.1 cells or AT2.1 vector(AT2.1N) controls and approach AT3.1 cell neuropilin-1 levels andmigration activity.

FIG. 19 shows expression of NP-1, NP-2 and β-actin in cancer cell linesand endothelial cells using reverse transcriptase PCR following primers:

Human NP-1 Forward (328-351): 5′ TTTCGCAACGATAAATGTGGCGAT 3′(SEQ ID NO: 7) Reverse (738-719): 5′ TATCACTCCACTAGGTGTTG 3′(SEQ ID NO: 8) Human NP-2 Forward (513-532): 5′ CCAACCAGAAGATTGTCCTC 3′(SEQ ID NO: 9) Reverse (1181-1162): 5′ GTAGGTAGATGAGGCACTGA 3′.(SEQ ID NO: 10)

FIG. 20 shows the effects of collapsin-1 treatment on PAE cell motilityin a Boyden chamber. Collapsin-1 inhibits, by about 65% the basalmigration of PAE cells expressing neuropilin-1 but not PAE cellsexpressing KDR. alone One collapsin unit is about 3 ng/ml.

FIGS. 21A and 21B show results of the aortic ring assay. Collapsin wasadded (FIG. 21A) or not added (FIG. 21B) to a segment of rat aortic ringand the migration of endothelial cells out of the rings and theirformation of tubes was monitored after a week in organ culture.Migration and tube formation are inhibited by collapsin-1.

DETAILED DESCRIPTION OF THE INVENTION

We have discovered that there are VEGF receptors (VEGFR) and neuropilinssuch as VEGF₁₆₅R/NP-1 and NP-2 that are associated with metastaticpotential of a malignant cell and angiogenesis. As used herein,“neuropilin” includes not only VEGF₁₆₅R/NP-1 and NP-2 but any neuropilinor VEGFR, where the constituents share at least about 85% homology witheither of the above VEGF₁₆₅R/NP-1 and NP-2 can be used. More preferably,such constituent shares at least 90% homology. Still more preferably,each constituent shares at least 95% homology.

Homology is measured by means well known in the art. For example %homology can be determined by any standard algorithm used to comparehomologies. These include, but are not limited to BLAST 2.0 such asBLAST 2.0.4 and i. 2.0.5 available from the NIH (Seewww.ncbi.nlm.nkh.gov/BLAST/newblast.html) (Altschul, S. F., et al.Nucleic Acids Res. 25: 3389-3402 (1997)) and DNASIS (Hitachi SoftwareEngineering America, Ltd.). These programs should preferably be set toan automatic setting such as the standard default setting for homologycomparisons. As explained by the NIH, the scoring of gapped resultstends to be more biologically meaningful than ungapped results.

For ease of reference, this disclosure will generally talk aboutVEGF₁₆₅R/NP-1 and NP-2 and/or homologs thereof but all teaching areapplicable to the above-described homologs.

In another embodiment a VEGFR can be used as long as it binds to asequence having at least 90%, more preferably 95% homology to exon 7 ofVEGF₁₆₅. These VEGF receptors and neuropilins, e.g., VEGF₁₆₅R/NP-1 andNP-2, are associated with both tumor metastases and angiogenesis. Wehave shown that expression of VEGF₁₆₅R/NP-1 and NP-2 is upregulated inhighly metastatic prostate cancer cell lines relative to poorlymetastatic or nonmetastatic lines. Thus, expression of VEGF₁₆₅R/NP-1 andNP-2 is associated with a tumors metastatic potential.

In accordance with the present invention, antagonists of neuropilinreceptor function can be used inhibit or prevent the metastasis processand/or angiogenesis. Antagonists of the invention can block thereceptors preventing ligand binding, disrupt receptor function, orinhibit receptor occurrence. Specific antagonists include, for example,compounds that bind to NP-1 or NP-2 and antibodies that specificallybinds the receptor at a region that inhibits receptor function. Forexample, one can add an effective amount of a compound that binds toNP-1 to disrupt receptor fuction and thus inhibit metastasis.

Preferred antagonists include members of the semaphorin/collapsinsfamily. We have surprisingly discovered that members of thesemaphorin/collapsins family are not only inhibitors of neuronalguidance but also inhibitors of endothelial and tumor cell motility incells that express neuropilin. Collapsin-1 is a particularly preferredantagonist. Other members of the semaphorin collapsin family can beselected by screening for neuropilin binding.

Semaphorin/collapsins are a family of 100 kDa glycoproteins (Luo, et al.(1993) Cell 75: 217-2271 Kolodkin, et al., (1993) Cell 75: 1389-1399,Behar, et al., (1996) Nature 383: 525-528.) Semaphorins are themammalian homologue and collapsins are the chick homologue. Semaphorinsare expressed primarily in the developing CNS, but are also found indeveloping bones and heart. The receptors for the semaphorins areneuropilin-1 and neuropilin-2 (He, et al., Cell 90, 739-751 (1997),Kolodkin, et al, Cell 90, 753-762 (1997)) and there is ligand bindingspecificity for different semaphorin family members (Chen, et al.,Neuron 19:547-559 (1997)). The K_(d) for semaphorin binding is about3×10⁻¹° M, similar to that for VEGF₁₆₅ binding to neuropilin-1.Semaphorins mediate neuronal guidance by repelling and collapsingadvancing dorsal root ganglion (DRG) growth cones.

Semaphorin/collapsins are know in the art and can be isolated fromnatural sources or produced using recombinant DNA methods. See, forexample, U.S. Pat. No. 5,807,826. Additionally, fragments of thesemaphorin/collapsins may be used. For example, a 70 amino acid regionwithin the semaphorin domain specifies the biological activities ofthree collapsin family members (Koppel, et al., Neuron 19: 531-537).

Pure recombinant chick collapsin-1 (semaphorin III) was can be producedby the methods set forth in the following references (Luo, et al. (1993)Cell 75: 217-227.); Koppel, et al. J. Biol. Chem. 273: 15708-15713,Feiner, et al. (1997) Neuron 19: 539-545).

We have shown that when collapsin-1 was added to cultures of porcineendothelial cells (PAE) and PAE neuropilin-1 and/or KDR transfectants,¹²⁵I-Collapsin was found to bind to PAE cells expressing neuropilin-1but not to PAE cells expressing KDR. Furthermore, in a Boyden chamberassay, collapsin-1 inhibited the basal migration of PAE expressingneuropilin-1, by about 60-70%, but had no effect on parental PAE or PAEexpressing KDR alone (FIG. 20). Inhibition was dose-dependent andhalf-maximal inhibition occurred with 50 collapsing units/ml (asmeasured on DRG, 1 CU=3 ng/ml). Thus, semaphorin/collapsins inhibit themotility of non-neuronal cells as long as neuropilin-1 is expressed.

Antibodies that specifically binds the NP at a region that inhibitsreceptor function can also be used as antagonists of the invention.Antibodies may be raised against either a peptide of the receptor or thewhole molecule. Such a peptide may be presented together with a carrierprotein, such as an KLH, to an animal system or, if it is long enough,say 25 amino acid residues, without a carrier.

In accordance with yet another aspect of the present invention, thereare provided isolated antibodies or antibody fragments which selectivelybinds the receptor. The antibody fragments include, for example, Fab,Fab′, F(ab′)2 or Fv fragments. The antibody may be a single chainantibody, a humanized antibody or a chimeric antibody.

Antibodies, or their equivalents, or other receptor antagonists may alsobe used in accordance with the present invention for the treatment orprophylaxis of cancers. Administration of a suitable dose of theantibody or the antagonist may serve to block the receptor and this mayprovide a crucial time window in which to treat the malignant growth.

Prophylaxis may be appropriate even at very early stages of the disease,as it is not known what specific event actually triggers metastasis inany given case. Thus, administration of the antagonists which interferewith receptor activity, may be effected as soon as cancer is diagnosed,and treatment continued for as long as is necessary, preferably untilthe threat of the disease has been removed. Such treatment may also beused prophylactically in individuals at high risk for development ofcertain cancers, e.g., prostate or breast.

It will be appreciated that antibodies for use in accordance with thepresent invention may be monoclonal or polyclonal as appropriate.Antibody equivalents of these may comprise: the Fab′ fragments of theantibodies, such as Fab, Fab′, F(ab′)₂ and Fv; idiotopes; or the resultsof allotope grafting (where the recognition region of an animal antibodyis grafted into the appropriate region of a human antibody to avoid animmune response in the patient), for example. Single chain antibodiesmay also be used. Other suitable modifications and/or agents will beapparent to those skilled in the art.

Chimeric and humanized antibodies are also within the scope of theinvention. It is expected that chimeric and humanized antibodies wouldbe less immunogenic in a human subject than the correspondingnon-chimeric antibody. A variety of approaches for making chimericantibodies, comprising for example a non-human variable region and ahuman constant region, have been described. See, for example, Morrisonet al., Proc. Natl. Acad. Sci. U.S.A. 81,6851 (1985); Takeda, et al.,Nature 314,452 (1985), Cabilly et al., U.S. Pat. No. 4,816,567; Boss etal., U.S. Pat. No. 4,816,397; Tanaguchi et al., European PatentPublication EP 171496; European Patent Publication 0173494, UnitedKingdom Patent GB 2177096B. Additionally, a chimeric antibody can befurther “humanized” such that parts of the variable regions, especiallythe conserved framework regions of the antigen-binding domain, are ofhuman origin and only the hypervariable regions are of non-human origin.Such altered immunoglobulin molecules may be made by any of severaltechniques known in the art, (e.g., Teng et al., Proc. Natl. Acad. Sci.U.S.A., 80, 7308-7312 (1983); Kozbor et al., Immunology Today, 4, 7279(1983); Olsson et al., Meth. Enzymol., 92, 3-16 (1982)), and arepreferably made according to the teachings of PCT Publication WO92/06193or EP 0239400. Humanized antibodies can be commercially produced by, forexample, Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, GreatBritain.

The present invention further provides use of neuropilin forintracellular or extracellular targets to affect binding. Intracellulartargeting can be accomplished through the use of intracellularlyexpressed antibodies referred to as intrabodies. Extracellular targetingcan be accomplished through the use of receptor specific antibodies.

These methods can be used to inhibit metastasis in malignant cells as wehave found that the presence of these receptors is positively correlatedwith metastasis. One can treat a range of afflictions or diseasesassociated with expression of the receptor by directly blocking thereceptor. This can be accomplished by a range of different approaches.One preferred approach is the use of antibodies that specifically blockVEGF binding to the receptor. For example, an antibody to the VEGFbinding site. Antibodies to these receptors can be prepared by standardmeans. For example, one can use single chain antibodies to target thesebinding sites.

The antibody can be administered by a number of methods. One preferredmethod is set forth by Marasco and Haseltine in PCT WO94/02610, which isincorporated herein by reference. This method discloses theintracellular delivery of a gene encoding the antibody. One wouldpreferably use a gene encoding a single chain antibody. The antibodywould preferably contain a nuclear localization sequence. One preferablyuses an SV40 nuclear localization signal. By this method one canintracellularly express an antibody, which can block VEGF₁₆₅R/NP-1 orNP-2 functioning in desired cells.

DNA encoding human VEGF₁₆₅R/NP-1 or NP-2 and recombinant humanVEGF₁₆₅R/NP-1 or NP-2 may be produced according to the methods set forthin the Examples.

The receptors are preferably produced by recombinant methods. A widevariety of molecular and biochemical methods are available forgenerating and expressing the polypeptides of the present invention; seee.g. the procedures disclosed in Molecular Cloning, A Laboratory Manual(2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor), CurrentProtocols in Molecular Biology (Eds. Aufubel, Brent, Kingston, More,Feidman, Smith and Stuhl, Greene Publ. Assoc., Wiley-Interscience, NY,N.Y. 1992) or other procedures that are otherwise known in the art. Forexample, the polypeptides of the invention may be obtained by chemicalsynthesis, expression in bacteria such as E. coli and eukaryotes such asyeast, baculovirus, or mammalian cell-based expression systems, etc.,depending on the size, nature and quantity of the polypeptide.

The term “isolated” means that the polypeptide is removed from itsoriginal environment (e.g., the native VEGF molecule). For example, anaturally-occurring polynucleotides or polypeptides present in a livinganimal is not isolated, but the same polynucleotides or DNA orpolypeptides, separated from some or all of the coexisting materials inthe natural system, is isolated. Such polynucleotides could be part of avector and/or such polynucleotides or polypeptides could be part of acomposition, and still be isolated in that such vector or composition isnot part of its natural environment.

Where it is desired to express the receptor or a fragment thereof, anysuitable system can be used. The general nature of suitable vectors,expression vectors and constructions therefor will be apparent to thoseskilled in the art.

Suitable expression vectors may be based on phages or plasmids, both ofwhich are generally host-specific, although these can often beengineered for other hosts. Other suitable vectors include cosmids andretroviruses, and any other vehicles, which may or may not be specificfor a given system. Control sequences, such as recognition, promoter,operator, inducer, terminator and other sequences essential and/oruseful in the regulation of expression, will be readily apparent tothose skilled in the art.

Correct preparation of nucleotide sequences may be confirmed, forexample, by the method of Sanger et al. (Proc. Natl. Acad. Sci. USA74:5463-7 (1977)).

A DNA fragment encoding the receptor or fragment thereof, may readily beinserted into a suitable vector. Ideally, the receiving vector hassuitable restriction sites for ease of insertion, but blunt-endligation, for example, may also be used, although this may lead touncertainty over reading frame and direction of insertion. In such aninstance, it is a matter of course to test transformants for expression,1 in 6 of which should have the correct reading frame. Suitable vectorsmay be selected as a matter of course by those skilled in the artaccording to the expression system desired.

By transforming a suitable organism or, preferably, eukaryotic cellline, such as HeLa, with the plasmid obtained, selecting thetransformant with ampicillin or by other suitable means if required, andadding tryptophan or other suitable promoter-inducer (such asindoleacrylic acid) if necessary, the desired polypeptide or protein maybe expressed. The extent of expression may be analyzed by SDSpolyacrylamide gel electrophoresis-SDS-PAGE (Lemelli, Nature 227:680-685(1970)).

Suitable methods for growing and transforming cultures etc. are usefullyillustrated in, for example, Maniatis (Molecular Cloning, A LaboratoryNotebook, Maniatis et al. (eds.), Cold Spring Harbor Labs, N.Y. (1989)).

Cultures useful for production of polypeptides or proteins may suitablybe cultures of any living cells, and may vary from prokaryoticexpression systems up to eukaryotic expression systems. One preferredprokaryotic system is that of E. coli, owing to its ease ofmanipulation. However, it is also possible to use a higher system, suchas a mammalian cell line, for expression of a eukaryotic protein.Currently preferred cell lines for transient expression are the HeLa andCos cell lines. Other expression systems include the Chinese HamsterOvary (CHO) cell line and the baculovirus system.

Other expression systems which may be employed include streptomycetes,for example, and yeasts, such as Saccharomyces spp., especially S.cerevisiae. Any system may be used as desired, generally depending onwhat is required by the operator. Suitable systems may also be used toamplify the genetic material, but it is generally convenient to use E.coli for this purpose when only proliferation of the DNA is required.

The polypeptides and proteins may be isolated from the fermentation orcell culture and purified using any of a variety of conventional methodsincluding: liquid chromatography such as normal or reversed phase, usingHPLC, FPLC and the like; affinity chromatography (such as with inorganicligands or monoclonal antibodies); size exclusion chromatography;immobilized metal chelate chromatography; gel electrophoresis; and thelike. One of skill in the art may select the most appropriate isolationand purification techniques without departing from the scope of thisinvention.

The present invention also provides binding assays using VEGF₁₆₅R/NP-1or NP-2 that permit the ready screening for compounds which affect thebinding of the receptor and its ligands, e.g., VEGF₁₆₅. These assays canbe used to identify compounds that modulate, preferably inhibitmetastasis and/or angiogenesis. However, it is also important to know ifa compound enhances metastasis so that its use can be avoided. Forexample, in a direct binding assay the compound of interest can be addedbefore or after the addition of the labeled ligand, e.g., VEGF₁₆₅, andthe effect of the compound on binding or cell motility or angiogenesiscan be determined by comparing the degree of binding in that situationagainst a base line standard with that ligand, not in the presence ofthe compound. The assay can be adapted depending upon precisely what isbeing tested.

The preferred technique for identifying molecules which bind to theneuropilin receptor utilizes a receptor attached to a solid phase, suchas the well of an assay plate.

The binding of the candidate molecules, which are optionally labeled(e.g., radiolabeled), to the immobilized receptor can be measured.Alternatively, competition for binding of a known, labeled receptorligand, such as I-¹²⁵ VEGF₁₆₅, can be measured. For screening forantagonists, the receptor can be exposed to a receptor ligand, e.g.,VEGF₁₆₅, followed by the putative antagonist, or the ligand andantagonist can be added to the receptor simultaneously, and the abilityof the antagonist to block receptor activation can be evaluated. Forexample, VEGF antagonist activity may also be determined by inhibitionof binding of labeled VEGF₁₆₅ to VEGF₁₆₅R as disclosed in the Examples.

The ability of discovered antagonists to influence angiogenesis ormetastasis can also be determined using a number of know in vivo and invitro assays. Such assays are disclosed in Jain et al., Nature Medicine3, 1203-1208 (1997), and the examples.

Where the present invention provides for the administration of, forexample, antibodies to a patient, then this may be by any suitableroute. If the tumor is still thought to be, or diagnosed as, localized,then an appropriate method of administration may be by injection directto the site. Administration may also be by injection, includingsubcutaneous, intramuscular, intravenous and intradermal injections.

Formulations may be any that are appropriate to the route ofadministration, and will be apparent to those skilled in the art. Theformulations may contain a suitable carrier, such as saline, and mayalso comprise bulking agents, other medicinal preparations, adjuvantsand any other suitable pharmaceutical ingredients. Catheters are onepreferred mode of administration.

Neuropilin expression may also be inhibited in vivo by the use ofantisense technology. Antisense technology can be used to control geneexpression through triple-helix formation or antisense DNA or RNA, bothof which methods are based on binding of a polynucleotide to DNA or RNA.An antisense nucleic acid molecule which is complementary to a nucleicacid molecule encoding receptor can be designed based upon the isolatednucleic acid molecules encoding the receptor provided by the invention.An antisense nucleic acid molecule can comprise a nucleotide sequencewhich is complementary to a coding strand of a nucleic acid, e.g.complementary to an mRNA sequence, constructed according to the rules ofWatson and Crick base pairing, and can hydrogen bond to the codingstrand of the nucleic acid. The antisense sequence complementary to asequence of an mRNA can be complementary to a sequence in the codingregion of the mRNA or can be complementary to a 5′ or 3′ untranslatedregion of the mRNA. Furthermore, an antisense nucleic acid can becomplementary in sequence to a regulatory region of the gene encodingthe mRNA, for instance a transcription initiation sequence or regulatoryelement. Preferably, an antisense nucleic acid complementary to a regionpreceding or spanning the initiation codon or in the 3′ untranslatedregion of an mRNA is used. An antisense nucleic acid can be designedbased upon the nucleotide sequence shown in SEQ ID NO:1 (VEGF₁₆₅R/NP-1)or SEQ ID NO:3 (NP-2). A nucleic acid is designed which has a sequencecomplementary to a sequence of the coding or untranslated region of theshown nucleic acid. Alternatively, an antisense nucleic acid can bedesigned based upon sequences of a VEGF₁₆₅R gene, which can beidentified by screening a genomic DNA library with an isolated nucleicacid of the invention. For example, the sequence of an importantregulatory element can be determined by standard techniques and asequence which is antisense to the regulatory element can be designed.

The antisense nucleic acids and oligonucleotides of the invention can beconstructed using chemical synthesis and enzymatic ligation reactionsusing procedures known in the art. The antisense nucleic acid oroligonucleotide can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids e.g. phosphorothioate derivatives and acridine substitutednucleotides can be used. Alternatively, the antisense nucleic acids andoligonucleotides can be produced biologically using an expression vectorinto which a nucleic acid has been subcloned in an antisense orientation(i.e. nucleic acid transcribed from the inserted nucleic acid will be ofan antisense orientation to a target nucleic acid of interest). Theantisense expression vector is introduced into cells in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal., Antisense RNA as a molecular tool for genetic analysis,Reviews—Trends in Genetics, Vol. 1 (1) 1986.

The term “pharmaceutically acceptable” refers to compounds andcompositions which may be administered to mammals without unduetoxicity. Exemplary pharmaceutically acceptable salts include mineralacid salts such as hydrochlorides, hydrobromides, phosphates, sulfates,and the like; and the salts of organic acids such as acetates,propionates, malonates, benzoates, and the like.

The antagonists of the invention are administered orally, topically, orby parenteral means, including subcutaneous and intramuscular injection,implantation of sustained release depots, intravenous injection,intranasal administration, and the like. Accordingly, antagonists of theinvention may be administered as a pharmaceutical composition comprisingthe antibody or nucleic acid of the invention in combination with apharmaceutically acceptable carrier. Such compositions may be aqueoussolutions, emulsions, creams, ointments, suspensions, gels, liposomalsuspensions, and the like. Suitable carriers (excipients) include water,saline, Ringer's solution, dextrose solution, and solutions of ethanol,glucose, sucrose, dextran, mannose, mannitol, sorbitol, polyethyleneglycol (PEG), phosphate, acetate, gelatin, collagen, Carbopol™,vegetable oils, and the like. One may additionally include suitablepreservatives, stabilizers, antioxidants, antimicrobials, and bufferingagents, for example, BHA, BHT, citric acid, ascorbic acid, tetracycline,and the like. Cream or ointment bases useful in formulation includelanolin, Silvadene™ (Marion), Aquaphor™ (Duke Laboratories), and thelike. Other topical formulations include aerosols, bandages, and otherwound dressings. Alternatively one may incorporate or encapsulate thecompounds such as an antagonist in a suitable polymer matrix ormembrane, thus providing a sustained-release delivery device suitablefor implantation near the site to be treated locally. Other devicesinclude indwelling catheters and devices such as the Alzet™ minipump.Ophthalmic preparations may be formulated using commercially availablevehicles such as Sorbi-care™ (Allergan), Neodecadron™ (Merck, Sharp &Dohme), Lacrilube™, and the like, or may employ topical preparationssuch as that described in U.S. Pat. No. 5,124,155, incorporated hereinby reference. Further, one may provide an antagonist in solid form,especially as a lyophilized powder. Lyophilized formulations typicallycontain stabilizing and bulking agents, for example human serum albumin,sucrose, mannitol, and the like. A thorough discussion ofpharmaceutically acceptable excipients is available in Remington'sPharmaceutical Sciences (Mack Pub. Co.).

The NP antagonists of the invention can be combined with atherapeutically effective amount of another molecule which negativelyregulates angiogenesis which may be, but is not limited to, TNP-470,platelet factor 4, thrombospondin-1, tissue inhibitors ofmetalloproteases (TIMP1 and TIMP2), prolactin (16-Kd fragment),angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF solublereceptor, transforming growth factor beta, interferon alfa, soluble KDRand FLT-1 receptors and placental proliferin-related protein.

An NP antagonist of the invention may also be combined withchemotherapeutic agents.

The DNA encoding an antagonist, e.g., a collapsin, can be used in theform of gene therapy and delivered to a host by any method known tothose of skill in the art to treat disorders associated with VEGF.

The amount of an NP antagonist required to treat any particular disorderwill of course vary depending upon the nature and severity of thedisorder, the age and condition of the subject, and other factorsreadily determined by one of ordinary skill in the art.

All references cited above or below are herein incorporated byreference.

The present invention is further illustrated by the following Examples.These Examples are provided to aid in the understanding of the inventionand are not construed as a limitation thereof.

Example 1 Experimental Procedures Materials

Cell culture media, lipofectin and lipofectamin reagents fortransfection were purchased from Life Technologies. Human recombinantVEGF₁₆₅ and VEGF₁₂₁ were produced in Sf-21 insect cells infected withrecombinant baculovirus vectors encoding either human VEGF₁₆₅ or VEGF₁₂₁as previously described (Cohen et al., Growth Factors, 7, 131-138(1992); Cohen et al., J. Biol. Chem., 270, 11322-11326 (1995)). GST VEGFexons 7+8 fusion protein was prepared in E. Coli and purified aspreviously described (Soker et al., J. Biol. Chem., 271, 5761-5767(1996)). Heparin, hygromycin B and protease inhibitors were purchasedfrom Sigma (St. Louis, Mo.). ¹²⁵I-Sodium, 32P-dCTP, and GeneScreen-Plushybridization transfer membrane were purchased from DuPont NEN (Boston,Mass.). Disuccinimidyl suberate (DSS) and IODO-BEADS were purchased fromPierce Chemical Co. (Rockford, Ill.). Con A Sepharose was purchased fromPharmacia LKB Biotechnology Inc. (Piscataway, N.J.). RNAzol-B waspurchased from TEL-TEST Inc. (Friendswood, Tex.). Silver Stain kit andTrans-Blot PVDF membranes were purchased from Bio-Rad Laboratories(Hercules, Calif.). Multiple tissue northern blot membranes werepurchased from Clontech (Palo Alto, Calif.). PolyATract mRNA isolationkits were purchased from Promega (Madison, Wis.). RediPrime DNA labelingkits and molecular weight markers were purchased from Amersham(Arlington Heights, Ill.). Plasmids: pcDNA3.1 was purchased fromInvitrogen (Carlsbad, Calif.), and pCPhygro, containing the CMV promoterand encoding hygromycin B phosphorylase, was kindly provided by Dr.Urban Deutsch (Max Plank Institute, Bad Nauheim, Germany). Restrictionendonucleases and Ligase were purchased from New England Biolabs, Inc(Beverly, Mass.). NT-B2 photographic emulsion and x-ray film werepurchased from the Eastman Kodak company (Rochester N.Y.).

Cell Culture

Human umbilical vein EC (HUVEC) were obtained from American Type CultureCollection (ATCC) (Rockville, Md.), and grown on gelatin coated dishesin M-199 medium containing 20% fetal calf serum (FCS) and a mixture ofglutamine, penicillin and streptomycin (GPS). Basic FGF (2 ng/ml) wasadded to the culture medium every other day. Parental porcine aorticendothelial (PAE) cells and PAE cells expressing KDR (PAE/KDR)(Waltenberger et al., J. Biol. Chem. 269, 26988-26995 (1994)) werekindly provided by Dr. Lena Claesson-Welsh and were grown in F12 mediumcontaining 10% FCS and GPS. MDA-MB-231 cells and MDA-MB-453 cells wereobtained from ATCC, and grown in DMEM containing 10% FCS and GPS. Thehuman melanoma cell lines, RU-mel, EP-mel and WK-mel were kindlyprovided by Dr. Randolf Byer (Boston University Medical School, Boston,Mass.), and grown in DMEM containing 2% FCS, 8% calf serum and GPS.Human metastatic prostate adenocarcinoma, LNCaP and prostate carcinoma,PC3 cells were kindly provided by Dr. Michael Freeman (Children'sHospital, Boston, Mass.), and grown in RPMI 1640 containing 5% FCS andGPS.

Purification and Protein Sequencing

Approximately 5×10⁸ MDA-MB-231 cells grown in 150 cm dishes were washedwith PBS containing 5 mM EDTA, scraped and centrifuged for 5 min at 500g. The cell pellet was lysed with 150 ml of 20 mM HEPES, pH 8.0, 0.5%triton X-100 and protease inhibitors including 1 mM AEBSF, 5 μg/mlleupeptin and 5 μg/ml aprotinin for 30 min on ice, and the lysate wascentrifuged at 30,000×g for 30 min. MnCl₂ and CaCl₂ were added to thesupernatant to obtain a final concentration of 1 mM each. The lysate wasabsorbed onto a Con A Sepharose column (7 ml) and bound proteins wereeluted with 15 ml 20 mM HEPES, pH 8.0, 0.2 M NaCl, 0.1% triton X-100 and1 M methyl-α-D-mannopyranoside at 0.2 ml/min. The elution was repeatedtwice more at 30 minute intervals. The Con A column eluates were pooledand incubated for 12 h at 4° C. with 0.5 ml of VEGF₁₆₅-Sepharose beads,containing about 150 μg VEGF₁₆₅, prepared as described previously(Wilchek and Miron, Biochem. Int. 4, 629-635. (1982)). TheVEGF₁₆₅-Sepharose beads were washed with 50 ml of 20 mM HEPES, pH 8.0,0.2 M NaCl and 0.1% triton X-100 and then with 25 ml of 20 mM HEPES, pH8.0. The beads were boiled in SDS-PAGE buffer and bound proteins wereseparated by 6% SDS-PAGE. Proteins were transferred to a TransBlot PVDFmembrane using a semi-dry electric blotter (Hoeffer Scientific), and thePVDF membrane was stained with 0.1% Coomassie Brilliant Blue in 40%methanol. The two prominent proteins in a 130-140 kDa doublet were cutout separately and N-terminally sequenced using an Applied Biosystemsmodel 477A microsequenator as a service provided by Dr. William Lane ofthe Harvard Microchemistry facility (Cambridge, Mass.).

Expression Cloning and DNA Sequencing

Complementary DNA (cDNA) was synthesized from 5 μg 231 mRNA.Double-stranded cDNA was ligated to EcoRI adaptors, andsize-fractionated on a 5-20% potassium acetate gradient. DNA fragmentslarger than 2 kb were ligated to an eukaryotic expression plasmid,pcDNA3.1. The plasmid library was transfected into E. coli to yield aprimary library of approximately 1×10⁷ individual clones. A portion ofthe transformed bacteria was divided into 240 pools, each representingapproximately 3×10³ individual clones. DNA prepared from each pool wasused to transfect COS-7 cells seeded in 12 well dishes, using theLipofectin reagent according to the manufacturer's instructions. Threedays after transfection, the cells were incubated on ice for 2 h with¹²⁵I-VEGF₁₆₅ (10 ng/ml) in the presence of 1 μg/ml heparin, washed andfixed with 4% paraformaldehyde in PBS. ¹²⁵I-VEGF₁₆₅ binding toindividual cells was detected by overlaying the monolayers withphotographic emulsion, NT-B2, and developing the emulsion after two daysas described (Gearing et al., 1989). Seven positive DNA pools wereidentified and DNA from one of the positive pools was used to transformE. Coli. The E. coli were sub-divided into 50 separate pools and platedonto 50 LB ampicillin dishes, with each pool representing approximately100 clones. DNA made from these pools was transfected into COS-7 cellswhich were screened for ¹²⁵I-VEGF₁₆₅ binding as described above. Twentypositive pools were detected at this step, and their corresponding DNAswere used to transform E. Coli. Each pool was plated onto separate LBampicillin dishes and DNA was prepared from 96 individual colonies andscreened in a 96-well two dimensional grid for ¹²⁵I-VEGF₁₆₅ binding totransfected COS-7 cells as described above. Seven single clones wereidentified as being positive at this step. The seven positive plasmidclones were amplified and their DNA was analyzed by restriction enzymedigestion. Six clones showed an identical digestion pattern of digestand one was different. One clone from each group was submitted forautomated DNA sequencing.

Northern Analysis

Total RNA was prepared from cells in culture using RNAzol according tothe manufacturer's instructions. Samples of 20 μg RNA were separated ona 1% formaldehyde-agarose gel, and transferred to a GeneScreen-Plusmembrane. The membrane was hybridized with a ³²P labeled fragment ofhuman VEGF₁₆₅R/NP-1 cDNA, corresponding to nucleotides 63-454 in theORF, at 63° C. for 18 h. The membrane was washed and exposed to an x-rayfilm for 18 h. A commercially-obtained multiple human adult tissue mRNAblot (Clonetech, 2 μg/lane) was probed for human NP-1 in a similarmanner. The multiple tissue blot was stripped by boiling in the presenceof 0.5% SDS and re-probed with a ³²P labeled fragment of KDR cDNAcorresponding to nucleotides 2841-3251 of the ORF (Terman et al.,Oncogene 6, 1677-1683 (1991)).

Transfection of PAE Cells

Parental PAE cells and PAE cells expressing KDR (PAE/KDR) (Waltenbergeret al., 1994) were obtained from Dr. Lena Claesson-Welsh. Human NP-1cDNA was digested with XhoI and XbaI restriction enzymes and subclonedinto the corresponding sites of pCPhygro, to yield pCPhyg-NP-1. PAE andPAE/KDR cells were grown in 6 cm dishes and transfected with 5 μg ofpCPhyg-NP-1 using Lipofectamine, according to the manufacturer'sinstructions. Cells were allowed to grow for an additional 48 h and themedium was replaced with fresh medium containing 200 μg/ml hygromycin B.After 2 weeks, isolated colonies (5−10×10³ cell/colony) were transferredto separate wells of a 48 well dish and grown in the presence of 200/mlhygromycin B. Stable PAE cell clones expressing VEGF₁₆₅R/NP-1 (PAE/NP-1)or co-expressing VEGF₁₆₅R/NP-1 and KDR (PAE/KDR/NP-1) were screened forVEGF₁₆₅ receptor expression by binding and cross linking of¹²⁵I-VEGF₁₆₅. For transient transfection, PAE/KDR cells were transfectedwith VEGF₁₆₅R/NP-1 as described above and after three days ¹²⁵I-VEGF₁₆₅cross-linking analysis was carried out.

Radio-Iodination of VEGF, Binding and Cross-Linking Experiments.

The radio-iodination of VEGF₁₆₅ and VEGF_(in) using IODO-BEADS wascarried out as previously described (Soker et al., J. Biol. Chem. 272,31582-31588 (1997)). The specific activity ranged from 40,000-100,000cpm/ng protein. Binding and cross-linking experiments using ¹²⁵I-VEGF₁₆₅and ¹²⁵I-VEGF₁₂₁ were performed as previously described (Gitay-Goren etal., J. Biol. Chem. 267, 6093-6098 (1992); Soker et al., J. Biol. Chem.271, 5761-5767 (1996)). VEGF binding was quantitated by measuring thecell-associated radioactivity in a γ-counter (Beckman, Gamma 5500). Thecounts represent the average of three wells. All experiments wererepeated at least three times and similar results were obtained. Theresults of the binding experiments were analyzed by the method ofScatchard using the LIGAND program (Munson and Rodbard, 1980).¹²⁵I-VEGF₁₆₅ and ¹²⁵I-VEGF₁₂₁ cross linked complexes were resolved by 6%SDS/PAGE and the gels were exposed to an X-Ray film. X-ray films weresubsequently scanned by using an IS-1000 digital imaging system (AlphaInnotech Corporation)

Purification of VEGF₁₆₅R

Cross-linking of ¹²⁵I-VEGF₁₆₅ to cell surface receptors of 231 cellsresults in formation of a 165-175 kDa labeled complex (Soker et al., J.Biol. Chem. 271, 5761-5767 (1996)). These cells have about 1-2×10⁵VEGF₁₆₅ binding sites/cell. In contrast to VEGF₁₆₅, VEGF₁₂₁ does notbind to the 231 cells and does not form a ligand-receptor complex (Sokeret al., J. Biol. Chem. 271, 5761-5767 (1996)). The relatively highVEGF₁₆₅R number and the lack of any detectable KDR or Flt-1 mRNA in 231cells (not shown) suggested that these cells would be a useful sourcefor VEGF₁₆₅R purification. Preliminary characterization indicated thatVEGF₁₆₅R is a glycoprotein and accordingly, a 231 cell lysate preparedfrom approximately 5×10⁸ cells was absorbed onto a Con A Sepharosecolumn. Bound proteins, eluted from the Con A column, were incubatedwith VEGF₁₆₅-Sepharose and the VEGF₁₆₅-affinity purified proteins wereanalyzed by SDS-PAGE and silver staining (FIG. 9, lane 2). A prominentdoublet with a molecular mass of about 130-135 kDa was detected. Thissize is consistent with the formation of a 165-175 kDa complex of 40-45kDa VEGF₁₆₅ bound to receptors approximately 130-135 kDa in size (FIG.9, lane 1). The two bands were excised separately and N-terminal aminoacid sequencing was carried out (FIG. 1, right). Both the upper andlower bands had similar N-terminal amino acid sequences which showedhigh degrees of sequence homology to the predicted amino acid sequencesin the N-terminal regions of mouse (Kawakami et al., J. Neurobiol, 29,1-17 (1995)) and human neuropilin-1 (NP-1) (He and Tessier-Lavigne, Cell90739-751 (1997)).

Expression Cloning of VEGF₁₆₅R from 231 Cell-Derived mRNA

Concomitant with the purification, VEGF₁₆₅ R was cloned by expressioncloning (Aruffo and Seed, Proc. Natl. Acad. Sci. USA 84, 8573-8577(1987a); Aruffo and Seed, EMBO J., 6, 3313-3316 (1987b); Gearing et al.,EMBO J. 8, 3667-3676 (1989)). For expression cloning, 231 cell mRNA wasused to prepare a cDNA library of approximately 10⁷ clones in aeukaryotic expression plasmid. E. coli transformed with the plasmidlibrary were divided into pools. The DNA prepared from each pool weretransfected into COS-7 cells in separate wells and individual cells werescreened for the ability to bind ¹²⁵I-VEGF₁₆₅ as detected byautoradiography of monolayers overlayed with photographic emulsion (FIG.2A). After three rounds of subpooling and screening, seven singlepositive cDNA clones were obtained. FIG. 2B shows binding of¹²⁵I-VEGF₁₆₅ to COS-7 cells transfected with one of these singlepositive clones (clone A2).

Restriction enzyme analysis revealed that six of the seven positivesingle clones had identical restriction digestion patterns but that oneclone had a pattern that was different (not shown). Sequencing of one ofthese similar cDNA clones, clone A2 (FIG. 3), showed it to be identicalto a sequence derived from a human-expressed sequence tag data bank(dbEST). This sequence also showed a high percentage of homology to thesequence of mouse neuropilin, NP-1(Kawakami et al., J. Neurobiol 29,1-17 (1995)). After we had cloned human VEGF₁₆₅R, two groups reportedthe cloning of rat and human receptors for semaphorin III and identifiedthem to be NP-1 (He and Tessier-Lavigne, Cell 90, 739-751 (1997);Kolodkin et al., Cell 90, 753-762 (1997)). The 231 cell-derived VEGF₁₆₅RcDNA sequence is virtually identical (see figure legend 3 forexceptions) to the human NP-1 sequence (He and Tessier-Lavigne, Cell 90,739-751 (1997)). Significantly, the predicted amino acid sequenceobtained by expression cloning (FIG. 3) confirmed the identification ofVEGF₁₆₅R as NP-1 that was determined by N-terminal sequencing (FIG. 1),and we have therefore named this VEGF receptor, VEGF₁₆₅R/NP-1.

The human VEGF₁₆₅R/NP-1 cDNA sequence predicts an open reading frame(ORF) of 923 amino acids with two hydrophobic regions representingputative signal peptide and transmembrane domains (FIG. 3). Overall, thesequence predicts ectodomain, transmembrane and cytoplasmic domainsconsistent with the structure of a cell surface receptor. The N-terminalsequence obtained via protein purification as shown in FIG. 1 isdownstream of a 21 amino acid putative hydrophobic signal peptidedomain, thereby indicating directly where the signal peptide domain iscleaved and removed. The short cytoplasmic tail of 40 amino acids isconsistent with results demonstrating that soluble VEGF₁₆₅R/NP-1released by partial trypsin digestion of 231 cells is similar in size tointact VEGF₁₆₅R/NP-1 (not shown).

Sequence analysis of the one clone obtained by expression cloning thathad a different restriction enzyme profile predicted an open readingframe of 931 amino acids with about a 47% homology to VEGF₁₆₅R/NP-1(FIG. 4). This human cDNA has a 93% sequence homology with ratneuropilin-2 (NP-2) and is identical to the recently cloned human NP-2(Chen et al., Neuron, 19, 547-559 (1997)).

Expression of VEGF₁₆₅R/NP-1 in Adult Cell Lines and Tissues

Reports of NP-1 gene expression have been limited so far to the nervoussystem of the developing embryo (Takagi et al., Dev. Biol. 122, 90-100(1987); Kawakami et al., J. Neurobiol. 29, 1-17 (1995); Takagi et al.,Dev. Biol. 170, 207-222 (1995)). Cell surface VEGF₁₆₅R/NP-1, however, isassociated with non-neuronal adult cell types such as EC and a varietyof tumor-derived cells (Soker et al., J. Biol. Chem. 271, 5761-5767(1996)). Northern blot analysis was carried out to determine whethercells that crossed-linked ¹²⁵I-VEGF₁₆₅ also synthesized VEGF₁₆₅R/NP-1mRNA. (FIG. 5). VEGF₁₆₅R/NP-1 mRNA levels were highest in 231 and PC3cells. VEGF₁₆₅R/NP-1 mRNA was detected to a lesser degree in HUVEC,LNCaP, EP-mel and RU-mel cells. There was little if any expression inMDA-MB-453 and WK-mel cells. The VEGF₁₆₅R/NP-1 gene expression patternswere consistent with our previous results showing that HUVEC, 231, PC3,LNCaP, EP-mel and RU-mel cells bind ¹²⁵I-VEGF₁₆₅ to cell surfaceVEGF₁₆₅R/NP-1 but that MDA-MB-453 and WK-mel cells do not (Soker et al.,J. Biol. Chem. 271, 5761-5767 (1996)).

VEGF₁₆₅R/NP-1 gene expression was analyzed also by Northern blot in avariety of adult tissues in comparison to KDR gene expression (FIG. 6).VEGF₁₆₅R/NP-1 mRNA levels were relatively highly in adult heart andplacenta and relatively moderate in lung, liver, skeletal muscle, kidneyand pancreas. A relatively low level of VEGF₁₆₅R/NP-1 mRNA was detectedin adult brain. Interestingly, previous analysis of NP-1 gene expressionin mouse and chicken brain suggested that this gene was expressedprimarily during embryonic development and was greatly diminished afterbirth (Kawakami et al., J. Neurobiol. 29, 1-17 (1995); Takagi et al.,Dev. Biol. 170, 207-222 (1995)). The tissue distribution of KDR mRNA wassimilar to that of VEGF₁₆₅R/NP-1 with the exception that it was notexpressed highly in the heart. These results indicate that VEGF₁₆₅R/NP-1is expressed widely in adult non-neuronal tissue, including tissues inwhich angiogenesis occurs such as heart and placenta.

Characterization of VEGF₁₆₅ Binding to VEGF₁₆₅R/NP-1

In order to characterize the binding properties of VEGF₁₆₅R/NP-1,porcine aortic endothelial (PAE) cells were transfected with the cDNA ofVEGF₁₆₅R/NP-1. The PAE cells were chosen for these expression studiesbecause they express neither KDR, Flt-1 (Waltenberger et al., J. Biol.Chem. 269, 26988-26995 (1994)) nor VEGF₁₆₅R. Stable cell linessynthesizing VEGF₁₆₅R/NP-1 (PAE/NP-1) were established and ¹²⁵I-VEGF₁₆₅binding experiments were carried out (FIG. 7). ¹²⁵I-VEGF₁₆₅ binding toPAE/NP-1 cells increased in a dose-dependent manner and reachedsaturation at approximately 30 ng/ml demonstrating that VEGF₁₆₅R/NP-1 isa specific VEGF₁₆₅ receptor (FIG. 7A). Scatchard analysis of VEGF₁₆₅binding revealed a single class of VEGF₁₆₅ binding sites with a K_(d) ofapproximately 3.2×10⁻¹⁰ M and approximately 3×10⁵ ¹²⁵1-VEGF₁₆₅ bindingsites per cell (FIG. 7B). Similar K_(d) values were obtained for severalindependently-generated PAE/NP-1 clones, although the receptor numbervaried from clone to clone (not shown). The K_(d) of 3×10⁻¹⁰ M for thePAE/NP-1 cell lines is consistent with the 2-2.8×10⁻¹⁰ M K_(d) valuesobtained for VEGF₁₆₅R/NP-1 expressed naturally by HUVEC and 231 cells(Gitay-Goren et al., J. Biol. Chem. 267, 6093-6098 (1992); Soker et al.,J. Biol. Chem. 271, 5761-5767 (1996)). The binding of ¹²⁵I-VEGF₁₆₅ toPAE/NP-1 cells was enhanced by 1 μg/ml heparin (not shown), consistentwith previous studies showing that heparin enhances ¹²⁵I-VEGF₁₆₅ bindingto VEGF₁₆₅R/NP-1 on HUVEC and 231 cells (Gitay-Goren et al., J. Biol.Chem. 267, 6093-6098 (1992); Soker et al., J. Biol. Chem. 271, 5761-5767(1996)).

Isoform-Specific Binding of VEGF to Cells Expressing VEGF₁₆₅R/NP-1

VEGF₁₆₅, but not VEGF₁₂₁, binds to VEGF₁₆₅R/NP-1 on HUVEC and 231 cells(Gitay-Goren et al., J. Biol. Chem. 271, 5519-5523 (1992); Soker et al.,J. Biol. Chem. 271, 5761-5767 (1996)). To ascertain whether cellstransfected with VEGF₁₆₅R/NP-1 had the same binding specificity,PAE/NP-1 cells were incubated with ¹²⁵I-VEGF₁₆₅ or ¹²⁵I-VEGF₁₂₁ followedby cross-linking (FIG. 8). ¹²⁵I-VEGF₁₆₅ did not bind to parental PAEcells (FIG. 8, lane 3) but did bind to PAE/NP-1 cells via VEGF₁₆₅R/NP-1(FIG. 8, lane 4). The radiolabeled complexes formed with VEGF₁₆₅R/NP-1were similar in size to those formed in HUVEC (FIG. 8, lane 1) and PC3cells (FIG. 8, lane 2). On the other hand, ¹²⁵I-VEGF₁₂₁, did not bind toeither parental PAE (FIG. 8, lane 7) or to PAE/NP-1 cells (FIG. 8, lane8). These results demonstrate that the VEGF isoform-specific bindingthat occurs with cells expressing endogenous VEGF₁₆₅R/NP-1 such asHUVEC, 231 and PC3 cells, can be replicated in cells transfected withVEGF₁₆₅R/NP-1 cDNA and support the finding that VEGF₁₆₅R and NP-1 areidentical.

Co-Expression of VEGF₁₆₅R/NP-1 and KDR Modulates VEGF₁₆₅ Binding to KDR

To determine whether expression of VEGF₁₆₅R/NP-1 had any effect onVEGF₁₆₅ interactions with KDR, PAE cells that were previouslytransfected with KDR cDNA to produce stable clones of PAE/KDR cells(Waltenberger et al., J. Biol. Chem. 269, 26988-26995 (1994)), weretransfected with VEGF₁₆₅R/NP-1 cDNA and stable clones expressing bothreceptors (PAE/KDR/NP-1) were obtained. These cells bound ¹²⁵I-VEGF₁₆₅to KDR (FIG. 8, lane 6, upper complex) and to VEGF₁₆₅R/NP-1 (FIG. 8,lane 6, lower complex) to yield a cross-linking profile similar to HUVEC(FIG. 8, lane 1). On the other hand, the PAE/KDR/NP-1 cells bound¹²⁵I-VEGF₁₂₁ to form a complex only with KDR (FIG. 8, lanes 9 and 10),consistent with the inability of VEGF₁₂₁ to bind VEGF₁₆₅R/NP-1.

It appeared that in cells co-expressing KDR and VEGF₁₆₅R/NP-1 (FIG. 8,lane 6), the degree of ¹²⁵I-VEGF₁₆₅-KDR 240 kDa complex formation wasenhanced compared to the parental PAE/KDR cells (FIG. 8, lane 5). Theseresults were reproducible and the degree of ¹²⁵I-VEGF₁₆₅-KDR 240 kDacomplex formation in different clones was correlated positively with thelevels of VEGF₁₆₅R/NP-1 expressed (not shown). However, it could not beruled out definitively that these differential KDR binding results werepossibly due to clonal selection post-transfection. Therefore, parentalPAE/KDR cells were transfected with VEGF₁₆₅R/NP-1 cDNA and ¹²⁵I-VEGF₁₆₅was bound and cross-linked to the cells three days later in order toavoid any diversity of KDR expression among individual clones (FIG. 9).A labeled 240 kDa complex containing KDR was formed in parental PAE/KDRcells (FIG. 9, lane 1) and in PAE/KDR cells transfected with theexpression vector (FIG. 9, lane 2). However, when ¹²⁵I-VEGF₁₆₅ wascross-linked to PAE/KDR cells transiently expressing VEGF₁₆₅R/NP-1, amore intensely labeled 240 kDa complex, about 4 times greater, wasobserved (FIG. 9, lane 3), compared to parental PAE/KDR cells (FIG. 9,lane 1) and PAE/KDR cells transfected with expression vector (FIG. 9,lane 2). These results suggest that co-expression of KDR andVEGF₁₆₅R/NP-1 genes in the same cell enhances the ability of VEGF₁₆₅ tobind to KDR.

A GST-VEGF Exon 7+8 Fusion Protein Inhibits VEGF₁₆₅ Binding toVEGF₁₆₅R/NP-1 and KDR

We have shown that ¹²⁵I-VEGF₁₆₅ binds to VEGF₁₆₅R/NP-1 through its exon7-encoded domain (Soker et al., J. Biol. Chem. 271, 5761-5767 (1996)).In addition, a GST fusion protein containing the peptide encoded by VEGFexon 7+8 (GST-Ex 7+8), inhibits completely the binding of ¹²⁵I-VEGF₁₆₅to VEGF₁₆₅R/NP-1 associated with 231 cells and HUVEC (Soker et al., J.Biol. Chem. 271, 5761-5767 (1996); Soker et al., J. Biol. Chem. 272,31582-31588 (1997)). When, added to PAE/NP-1 cells, the fusion proteincompletely inhibited binding to VEGF₁₆₅R/NP-1 (FIG. 10, lane 2 comparedto lane 1). On the other hand, it did not inhibit ¹²⁵I-VEGF₁₆₅ bindingat all to KDR (FIG. 10, lane 4 compared to lane 3). Thus, these resultsdemonstrate that GST-Ex 7+8 binds directly to VEGF₁₆₅R/NP-1 but does notbind to KDR. The effects of GST-Ex 7+8 are different, however, in cellsco-expressing both VEGF₁₆₅R/NP-1 and KDR (PAE/KDR/NP-1). Consistent withthe results in FIGS. 8 and 9, the degree of ¹²⁵I-VEGF₁₆₅ binding to KDRin PAE/KDR/NP-1 cells (FIG. 10, lane 5) was greater than to the parentalPAE/KDR cells (FIG. 10, lane 3). Interestingly, in PAE/KDR/NP-1 cells,GST-Ex 7+8 inhibited not only ¹²⁵I-VEGF₁₆₅ binding to VEGF₁₆₅R/NP-1completely as expected, but it also inhibited binding to KDRsubstantially which was unexpected (FIG. 10, lane 6 compared to lane 5).In the presence of GST-Ex 7+8, binding of ¹²⁵I-VEGF₁₆₅ to KDR in thesecells was reduced to the levels seen in parental PAE/KDR cells notexpressing VEGF₁₆₅R/NP-1 (FIG. 10, lane 6 compared to lanes 3 and 4).Since the fusion protein does not bind directly to KDR, these resultssuggest that inhibiting the binding of ¹²⁵I-VEGF₁₆₅ to VEGF₁₆₅R/NP-1directly, inhibits its binding to KDR indirectly. Taken together, theresults in FIGS. 8, 9 and 10 suggest that interactions of VEGF₁₆₅ withVEGF₁₆₅R/NP-1 enhance VEGF interactions with KDR.

Neuropilin-1 is an Isoform-Specific VEGF₁₆₅ Receptor

Recently, we described a novel 130-135 kDa VEGF cell surface receptorthat binds VEGF₁₆₅ but not VEGF₁₂₁ and that we named, accordingly,VEGF₁₆₅R (Soker et al., J. Biol. Chem. 271, 5761-5767 (1996)). We havenow purified VEGF₁₆₅R, expression cloned its cDNA, and shown it to beidentical to human neuropilin-1 (NP-1) (He and Tessier-Lavigne, Cell 90739-751 (1997)). The evidence that VEGF₁₆₅R is identical to NP-1 andthat NP-1 serves as a receptor for VEGF₁₆₅ is as follows: i)purification of VEGF₁₆₅R protein from human MDA-MB-231 (231) cells usingVEGF affinity, yielded a 130-140 kDa doublet upon SDS-PAGE and silverstain. N-terminal sequencing of both proteins yielded the sameN-terminal sequence of 18 amino acids that demonstrated a high degree ofhomology to mouse NP-1 (Kawakami et al., J. Neurobiol. 29, 1-17 (1995));ii) After we purified VEGF₁₆₅R from human 231 cells, the cloning ofhuman NP-1 was reported (He and Tessier-Lavigne, Cell 90, 739-751(1997)) and the N-terminal sequence of human VEGF₁₆₅R was found to beidentical to a sequence in the N-terminal region of human NP-1; iii)Expression cloning using a 231 cell cDNA library resulted in isolationof several cDNA clones and their sequences were identical to the humanNP-1 cDNA sequence (He and Tessier-Lavigne, Cell 90, 739-751 (1997)).The combination of purification and expression cloning has the advantageover previous studies where only expression cloning was used (He andTessier-Lavigne, Cell 90, 739-751 (1997); Kolodkin et al., Cell 90,753-762 (1997)), in allowing unambiguous identification of the NP-1protein N-terminus; iv) Northern blot analysis of NP-1 gene expressionwas consistent with previous ¹²⁵I-VEGF₁₆₅ cross-linking experiments(Soker et al., J. Biol. Chem. 271, 5761-5767 (1996)). Cells that boundVEGF₁₆₅ to VEGF₁₆₅R synthesized relatively abundant NP-1 mRNA whilecells that showed very little if any VEGF₁₆₅ binding, did not synthesizemuch if any NP-1 mRNA; v) when NP-1 was expressed in PAE cells, thetransfected, but not the parental cells, were able to bind VEGF₁₆₅ butnot VEGF₁₂₁, consistent with the isoform specificity of bindingpreviously shown for HUVEC and 231 cells (Soker et al., J. Biol. Chem.271, 5761-5767 (1996)). Furthermore, the K_(d) of ¹²⁵I-VEGF₁₆₅ bindingof to PAE expressing NP-1 was about 3×10⁻¹⁰ M, consistent with previousK_(d) binding values of 2−2.8×10⁻¹° M for 231 cells and HUVEC (Soker etal., J. Biol. Chem. 271, 5761-5767 (1996)); and vi) The binding ofVEGF₁₆₅ to cells expressing NP-1 post-transfection was more efficient inthe presence of heparin as was the binding of this ligand to HUVEC and231 cells (Gitay-Goren et al., J. Biol. Chem. 267. 6093-6098 (1992);Soker et al., J. Biol. Chem. 271, 5761-5767 (1996)). Taken together,these results show not only that VEGF₁₆₅R is identical to NP-1 but thatit is a functional receptor that binds VEGF₁₆₅ in an isoform-specificmanner. Accordingly, we have named this VEGF receptor VEGF₁₆₅R/NP-1.

In addition to the expression cloning of VEGF₁₆₅R/NP-1 cDNA, anotherhuman cDNA clone was isolated whose predicted amino acid sequence was47% homologous to that of VEGF₁₆₅R/NP-1 and over 90% homologous to ratneuropilin-2 (NP-2) which was recently cloned (Kolodkin et al., Cell 90,753-762 (1997)). NP-2 binds members of the collapsin/semaphorin familyselectively (Chen et al., Neuron 19, 547-559 (1997)).

The discovery that NP-1 serves as a receptor for VEGF₁₆₅ was a surprisesince NP-1 had previously been shown to be associated solely with thenervous system during embryonic development (Kawakami et al., J.Neurobiol. 29, 1-17 (1995); Takagi et al., Dev. Biol. 170, 207-222(1995)) and more recently as a receptor for members of thecollapsin/semaphorin family (He and Tessier-Lavigne, Cell 90739-751(1997); Kolodkin et al., Cell 90, 753-762 (1997)). NP-1 is a 130-140 kDatransmembrane glycoprotein first identified in the developing Xenopusoptic system (Takagi et al., Dev. Biol. 122, 90-100 (1987); Takagi etal., Neuron 7, 295-307 (1991)). NP-1 expression in the nervous system ishighly regulated spatially and temporally during development and inparticular is associated with those developmental stages when axons areactively growing to form neuronal connections. (Fujisawa et al., Dev.Neurosci. 17, 343-349 (1995); Kawakami et al., J. Neurobiol 29, 1-17(1995); Takagi et al., Dev. Biol. 170, 207-222 (1995)). The NP-1 proteinis associated with neuronal axons but not the stomata (Kawakami et al.,J. Neurobiol 29, 1-17 (1995)). Functionally, neuropilin has been shownto promote neurite outgrowth of optic nerve fibers in vitro (Hirata etal., Neurosci. Res. 17, 159-169 (1993)) and to promote cell adhesiveness(Tagaki et al., Dev. Biol. 170, 207-222 (1995)). Targeted disruption ofNP-1 results in severe abnormalities in the trajectory of efferentfibers of the peripheral nervous system (Kitsukawa et al., Neuron 19,995-1005 (1997)). Based on the these studies, it has been suggested thatNP-1 is a neuronal cell recognition molecule that plays a role in axongrowth and guidance (Kawakami et al., J. Neurobiol. 29, 1-17 (1995); Heand Tessier-Lavigne, Cell 90, 739-751 (1997); Kitsukawa et al., Neuron19, 995-1005 1997; Kolodkin et al., Cell 90, 753-762 (1997)).

Our results are the first to show that VEGF₁₆₅R/NP-1 is also expressedin adult tissues, in contrast to the earlier studies that have shownthat NP-1 expression in Xenopus, chicken and mouse is limited to thedevelopmental and early post-natal stages (Fujisawa et al., Dev.Neurosci. 17, 343-349 (1995); Kawakami et al., J. Neurobiol. 29, 1-17(1995); Takagi et al., Dev. Biol. 170, 207-222 (1995)). For example, inmice, NP-1 is expressed in the developing nervous system starting in thedorsal root ganglia at day 9 and ceases at day 15 (Kawakami et al., J.Neurobiol. 29, 1-17 (1995). Our Northern blot analysis of human adulttissue demonstrates relatively high levels of VEGF₁₆₅R/NP-1 mRNAtranscripts in heart, placenta, lung, liver, skeletal muscle, kidney andpancreas. Interestingly, there is very little relative expression inadult brain, consistent with the mouse nervous system expression studies(Kawakami et al., J. Neurobiol. 29, 1-17 (1995)). VEGF₁₆₅R/NP-1 is alsoexpressed in a number of cultured non-neuronal cell lines including ECand a variety of tumor-derived cells. A possible function ofVEGF₁₆₅R/NP-1 in these cells is to mediate angiogenesis as will bediscussed below.

In addition, NP-1 has been identified as a receptor for thecollapsin/semaphorin family by expression cloning of a cDNA libraryobtained from rat E14 spinal cord and dorsal root ganglion (DRG) tissue(He and Tessier-Lavigne, Cell 90, 739-751 (1997); Kolodkin et al., Cell90, 753-762 (1997)). The collapsin/semaphorins (collapsin-D-1/SemaIII/Sem D) comprise a large family of transmembrane and secretedglycoproteins that function in repulsive growth cone and axon guidance(Kolodkin et al., Cell 75, 1389-1399 (1993)). The repulsive effect ofsema III for DRG cells was blocked by anti-NP-1 antibodies (He andTessier-Lavigne, Cell 90, 739-751 (1997); Kolodkin et al., Cell 90,753-762 (1997)). The K_(d) of sema III binding to NP-1, 0.15-3.25×10⁻¹°M (He and Tessier-Lavigne, Cell 90, 739-751 (1997); Kolodkin et al.,Cell 90, 753-762 (1997)) is similar to that of VEGF₁₆₅ bindingVEGF₁₆₅/NP-1, which is about 3×10⁻¹⁰ M. These results indicate that twostructurally different ligands with markedly different biologicalactivities, VEGF-induced stimulation of EC migration and proliferationon one hand, and sema III-induced chemorepulsion of neuronal cells, onthe other hand, bind to the same receptor and with similar affinity. Aninteresting question is whether the two ligands bind to the same site onVEGF₁₆₅R/NP-1 or to different sites. VEGF₁₆₅R/NP-1 has five discretedomains in its ectodomain, and it has been suggested that this diversityof protein modules in NP-1 is consistent with the possibility ofmultiple binding ligands for NP-1 (Takagi et al., Neuron 7, 295-307(1991); Feiner et al., Neuron 19 539-545 (1997); He and Tessier-Lavigne,Cell 90 739-751 (1997). Preliminary analysis does not indicate any largedegree of sequence homology between sema III and VEGF exon 7 which isresponsible for VEGF binding to VEGF₁₆₅R/NP-1 (Soker et al., J. Biol.Chem. 271, 5761-5767 (1996)). However there may be some 3-dimensionalstructural similarities between the two ligands. Since both neurons andblood vessels display branching and directional migration, the questionalso arises as to whether VEGF₁₆₅ displays any neuronal guidanceactivity and whether sema III has any EC growth factor activity. Thesepossibilities have not been examined yet. However, it may be that VEGFrequires two receptors, KDR and NP-1 for optimal EC growth factoractivity (Soker et al., J. Biol. Chem. 272, 31582-31588 (1997)) and thatsema III requires NP-1 and an as yet undetermined high affinity receptorfor optimal chemorepulsive activity (Feiner et al., Neuron 19, 539-545(1997) He and Tessier-Lavigne, Cell 90, 739-751 (1997); Kitsukawa etal., Neuron 19, 995-1005 (1997)), so that the presence of NP-1 alonemight not be sufficient for these ligands to display novel biologicalactivities. Future studies will determine whether there are anyconnections between the mechanisms that regulate neurogenesis andangiogenesis.

VEGF₁₆₅R/NP-1 Role Angiogenesis

VEGF₁₆₅R/NP-1 modulates the binding of VEGF₁₆₅ to KDR, a high affinityRTK that is an important regulator of angiogenesis as evidenced by KDRknock out experiments in mice (Shalaby et al., Nature 376, 62-66 (1995).The affinity of KDR for VEGF₁₆₅ is about 50 times greater than forVEGF₁₆₅R/NP-1 (Gitay-Goren et al., J. Biol. Chem. 287, 6003-6096 (1992);Waltenberger et al., J. Biol. Chem. 269, 26988-26995 (1994)). WhenVEGF₁₆₅R/NP-1 and KDR are co-expressed, the binding of ¹²⁵I-VEGF₁₆₅ toKDR is enhanced by about 4-fold compared to cells expressing KDR alone.The enhanced binding can be demonstrated in stable clones co-expressingVEGF₁₆₅R/NP-1 and KDR (PAE/KDR/NP-1 cells), and also in PAE/KDR cellstransfected transiently with VEGF₁₆₅R/NP-1 cDNA where clonal selectiondoes not take place. Conversely, when the binding of ¹²⁵I-VEGF₁₆₅ toVEGF₁₆₅R/NP-1 in PAE/KDR/NP-1 cells is inhibited completely by a GSTfusion protein containing VEGF exons 7+8 (GST-Ex 7+8), the binding toKDR is inhibited substantially, down to the levels observed in cellsexpressing KDR alone. The fusion protein binds to VEGF₁₆₅R/NP-1 directlybut is incapable of binding to KDR directly (Soker et al., J. Biol.Chem. 272, 31582-31588 (1997)). Although, not wishing to be bound bytheory, we believe that VEGF₁₆₅ binds to VEGF₁₆₅R/NP-1 via the exon7-encoded domain and facilitates VEGF₁₆₅ binding to KDR via the exon4-encoded domain (FIG. 11). VEGF₁₆₅R/NP-1, with its relatively highreceptor/cell number, about 0.2-2×10⁵ (Gitay-Goren et al., J. Biol.Chem. 287, 6003-6096 (1992); Soker et al., J. Biol. Chem. 271, 5761-5767(1996)), appears to serve to concentrate VEGF₁₆₅ on the cell surface,thereby providing greater access of VEGF₁₆₅ to KDR. Alternatively,binding to VEGF₁₆₅R/NP-1, VEGF₁₆₅ undergoes a conformational change thatenhances its binding to KDR. The end result would be elevated KDRsignaling and increased VEGF activity. Although we can demonstrateenhanced binding to KDR, to date we have not been able to demonstrateenhanced VEGF mitogenicity for PAE/KDR/NP-1 cells compared to PAE/KDRcells. One reason is that these cell lines do not proliferate readily inresponse to VEGF as do HUVEC (Waltenberger et al., J. Biol. Chem. 269,26988-26995 (1994). Nevertheless, we have shown that VEGF₁₆₅, whichbinds to both KDR and VEGF₁₆₅R/NP-1, is a better mitogen for HUVEC thanis VEGF₁₂₁, which binds only to KDR (Keyt et al., J. Biol. Chem. 271,5638-5646 (1996b); Soker et al., J. Biol. Chem. 272, 31582-31588 (1997).Furthermore, inhibiting VEGF₁₆₅ binding to VEGF₁₆₅R/NP-1 on HUVEC byGST-EX 7+8, inhibits binding to KDR and also inhibits VEGF₁₆₅-inducedHUVEC proliferation, down to the level induced by VEGF₁₂₁ (Soker et al.,J. Biol. Chem. 272, 31582-31588 (1997)). Taken together, these resultssuggest a role for VEGF₁₆₅R/NP-1 in mediating VEGF₁₆₅, but not VEGF₁₂₁mitogenic activity. The concept that dual receptors regulate growthfactor binding and activity has been previously demonstrated for TGF-β,bFGF and NGF (Lopez-Casillas et al., Cell 67, 785-795 (1991); Yayon etal., Cell 64, 841-848 (1991; Barbacid, Curr. Opin. Cell Biol. 7, 148-155(1995)).

Another connection between VEGF₁₆₅R/NP-1 and angiogenesis comes fromstudies in which NP-1 was overexpressed ectopically in transgenic mice(Kitsuskawa et al., Develop. 121, 4309-4318 (1995)). NP-1 overexpressionresulted in embryonic lethality and the mice died in utero no later thanon embryonic day 15.5 and those that survived the best had lower levelsof NP-1 expression. Mice overexpressing NP-1 displayed morphologicabnormalities in a limited number of non-neural tissues such as bloodvessels, the heart and the limbs. NP-1 was expressed in both the EC andin the mesenchymal cells surrounding the EC. The embryos possessedexcess and abnormal capillaries and blood vessels compared to normalcounterparts and in some cases dilated blood vessels as well. Some ofthe chimeric mice showed hemorrhaging, mainly in the head and neck.These results are consistent with the possibility that ectopicoverexpression of VEGF₁₆₅R/NP-1 results in inappropriate VEGF₁₆₅activity, thereby mediating enhanced and/or aberrant angiogenesis.Another piece of evidence for a link between NP-1 and angiogenesis comesfrom a recent report showing that in mice targeted for disruption of theNP-1 gene, the embryos have severe abnormalities in the peripheralnervous system but that their death in utero at days 10.5-12.5 is mostprobably due to anomalies in the cardiovascular system (Kitsukawa etal., Neuron 19, 995-1005 (1997)).

VEGF₁₆₅R/NP-1 is Associated with Tumor-Derived Cells

The greatest degree of VEGF₁₆₅R/NP-1 expression that we have detected sofar occurs in tumor-derived cells such as 231 breast carcinoma cells andPC3 prostate carcinoma cells, far more than occurs in HUVEC. The tumorcells express abundant levels of VEGF₁₆₅R/NP-1 mRNA and about 200,000VEGF₁₆₅ receptors/cell (Soker et al., J. Biol. Chem. 271, 5761-5767(1996)). On the other hand, these tumor cells do not express KDR orFlt-1 so that VEGF₁₆₅R/NP-1 is the only VEGF receptor associated withthese cells. The tumor cells are therefore useful for testing whetherVEGF₁₆₅R/NP-1 is a functional receptor for VEGF₁₆₅ in the absence of aKDR background. To date, we have not been able to show thatVEGF₁₆₅R/NP-1 mediates a VEGF₁₆₅ signal in tumor-derived cells asmeasured by receptor tyrosine phopshorylation. Nevertheless, VEGF₁₆₅might have an effect on tumor cells by inducing some, as yetundetermined activity such as enhanced survival, differentiation, ormotility. A recent report has demonstrated that glioma cells express a190 kDa protein that binds VEGF₁₆₅ but not VEGF₁₂₁ efficiently (Omura etal., J. Biol. Chem. 272, 23317-23322 (1997)). No stimulation of tyrosinephosphorylation could be demonstrated upon binding of VEGF₁₆₅ to thisreceptor. Whether the 190 kDa isoform-specific receptor is related toVEGF₁₆₅R/NP-1 is not known presently.

VEGF₁₆₅R/NP-1 may have a storage and sequestration function for VEGF₁₆₅.One might envision that VEGF₁₆₅ is produced by a tumor cell and binds toVEGF₁₆₅R/NP-1 on that cell via the exon 7-encoded domain (Soker et al.,J. Biol. Chem. 271, 5761-5767 (1996)). The stored VEGF₁₆₅ could be thenreleased to stimulate tumor angiogenesis in a paracrine manner.Alternatively, VEGF₁₆₅R/NP-1 may mediate a juxtacrine effect in whichVEGF₁₆₅ is bound to VEGF₁₆₅R/NP-1 on a tumor cell via the exon 7-encodeddomain and is also bound to KDR on a neighboring EC via the exon4-encoded domain (Keyt et al., J. Biol. Chem. 271, 5638-5646 (1996b)).Such a mechanism could result in a more efficient way for tumor cells toattract EC, thereby enhancing tumor angiogenesis.

In summary, we have demonstrated by independent purification andexpression cloning methods that the VEGF isoform specific receptor,VEGF₁₆₅R, is identical to NP-1, a cell surface protein previouslyidentified as playing a role in embryonic development of the nervoussystem and as being a receptor for the collapsins/semaphorins.Furthermore, binding to VEGF₁₆₅R/NP-1 enhances the binding of VEGF₁₆₅ toKDR on EC and tumor cells.

Experimental Rationale

We have discovered that tumor cell neuropilin-1 mediates tumor cellmotility and thereby metastasis. In a Boyden chamber motility assay,VEGF₁₆₅ (50 ng/ml) stimulates 231 breast carcinoma cell motility in adose-response manner, with a maximal 2-fold stimulation (FIG. 15A). Onthe other hand, VEGF₁₂₁ has no effect on motility of these cells (FIG.15B). Since 231 cells do not express KDR or Flt-1, these results suggestthat tumor cells are directly responsive to VEGF₁₆₅ and that VEGF₁₆₅might signal tumor cells via neuropilin-1. Possible candidates formediating VEGF₁₆₅-induced motility of carcinoma cells are PI3-kinase(PI3-K) (Carpenter, et al. (1996) Curr. Opin. Cell Biol. 8: 153-158.).Since 231 cells do not express KDR or Flt-1, these results suggest thattumor cells are directly responsive to VEGF₁₆₅ and that VEGF₁₆₅ mightsignal tumor cells via neuropilin-1.

The other type of evidence is that neuropilin-1 expression might beassociated with tumor cell motility. We have analyzed two variants ofDunning rat prostate carcinoma cells, AT2.1 cells, which are of lowmotility and low metastatic potential, and AT3.1 cells, which are highlymotile, and metastatic. Cross-linking and Northern blot analysis showthat AT3.1 cells express abundant neuropilin-1, capable of bindingVEGF₁₆₅, while AT2.1 cells don't express neuropilin-1 (FIG. 16).Immunostaining of tumor sections confirms the expression of neuropilin-1in AT3.1, but not AT2.1 tumors (FIG. 17). Furthermore, theimmunostaining shows that in subcutaneous AT3.1 and PC3 tumors, thetumor cells expressing neuropilin-1 are found preferentially at theinvading front of the tumor/dermis boundary (FIG. 17). To determine moredirectly whether neuropilin-1 expression is correlated with enhancedmotility, neuropilin-1 was overexpressed in AT2.1 cells (FIG. 18). Threestable clones of AT2.1 cells overexpressing neuropilin-1 had enhancedmotility in the Boyden chamber assay. These results indicate thatexpression of neuropilin-1 in AT2.1 cells enhances their motility. Takentogether, it appears that neuropilin-1 expression on tumor cells isassociated with the motile, metastatic phenotype.

Example 2 Experimental Procedures

1. Collapsin/semaphorins. Expression plasmids for expressing andpurifying His-tagged collapsin-1 from transfected 293T cells can beproduced according to the methods of (Koppel, et al. (1998) J. Biol.Chem. 273: 15708-15713, Feiner, et al. (1997) Neuron 19: 539-545.).Expression vectors for expressing sema E and sema IV alkaline phosphate(AP) conjugates in cells are disclosed in (He Z, Tessier-Lavigne M.(1997). Neuropilin is a receptor for the axonal chemorepellentsemaphorin III. Cell 90: 739-751.). Migration was measured in a Boydenchamber Falk, et al., J. Immunol. 118:239-247 (1980) with increasingconcentration of recombinant chick collapsin-1 in the bottom well andPAE cell transfectants in the upper well.2. Aortic Ring Assay. 200 gram rats were sacrificed and the aorta isdissected between the aortic arch and kidney artery and theadipofibrotic tissue around the aorta was removed. Aortic rings weresliced at 1 mm intervals and embedded in type I collagen gels. Each ringwas cultured in one well of a 48-well plate with serum-free endothelialcell medium (GIBCO). The number of microvessels were counted in eachring using a phase microscope (Miao, et al. (1997). J. Clin. Invest. 99:1565-1575.).

We established several endothelial cell lines by transfection ofparental porcine aortic endothelial cells (PAE), which normally do notexpress VEGF receptors (Soker, et al. (1998) Cell 92: 735-745). The celllines included PAE cells expressing neuropilin-1 alone (PAE/NP1), PAEcells expressing KDR alone (PAE/KDR) and PAE cells expressing bothneuropilin-1 and KDR (PAE/NP1/KDR). Collapsin-1 was obtained from Dr.Jon Raper, University of Pennsylvania (Luo, et al. (1993) Cell 75:217-227.).

Binding studies demonstrated that ¹²⁵I-collapsin-1 could bind to PAE/NP1cells and PAE/NP1/KDR cells but not at all to PAE or PAE/KDR cells. In aBoyden chamber assay, collapsin-1 at 50-100 collapsin units/ml (CU)inhibited the basal migration of PAE/NP and PAE/NP1/KDR cells by 70% buthad no inhibitory effect, whatsoever, on basal PAE or PAE/KDR cellmigration (FIG. 20). This effect is fairly potent since 1 CU representsabout 3 ng/ml protein. The collapsin-1 inhibitory effect was inhibitedby anti-neuropilin-1 antibodies. These results indicate that collapsinscan inhibit the migration of non-neuronal endothelial cells as long asthey express neuropilin-1.

Collapsin-1 inhibited the migration of PAE/NP and PAE/NP/KDR cells inthe presence of VEGF₁₆₅, to the same degree, the baseline being higher.We have also found that addition of collapsin in a rat aortic ring assay(a model for angiogenesis in vitro) inhibits the migration ofendothelial cells out of the ring, and endothelial tube formation (FIG.21).

The references cited throughout the specification are incorporatedherein by reference.

The present invention has been described with reference to specificembodiments. However, this application is intended to cover thosechanges and substitutions which may be made by those skilled in the artwithout departing from the spirit and the scope of the appended claims.

1. A method of inhibiting metastasis in a subject in need thereof, themethod comprising administering to a subject an effective amount of anantagonist of neuropilin-2 (NP-2).
 2. The method of claim 1, wherein theantagonist specifically binds NP-2.
 3. The method of claim 2, whereinthe antagonist specifically binds to an NP-2 having the sequence setforth in SEQ ID NO:4.
 4. The method of claim 1, wherein the antagonistis an antibody or antibody fragment thereof specifically binds NP-2. 5.The method of claim 4, wherein the antibody or antibody fragment thereofspecifically binds NP-2 at a VEGF binding site.
 6. The method of claim4, wherein the antibody or antibody fragment thereof specifically bindsNP-2 having the sequence set forth in SEQ ID NO:4.
 7. The method ofclaim 4, wherein the antibody or antibody fragment thereof ismonoclonal.
 8. The method of claim 4, wherein the antibody or antibodyfragment thereof is a humanized antibody or antibody fragment thereof.9. An isolated antibody or antibody fragment thereof that specificallybinds a neuropilin-2 (NP-2), wherein said isolated antibody specificallyinhibits binding of VEGF to NP-2.
 10. The isolated antibody or antibodyfragment thereof of claim 9, wherein said NP-2 has the sequence setforth in SEQ ID NO:4.